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ProteinLynx Global SERVER Version 2.2.5 User's Guide

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1. P08978 metal binding protein DHHC domain lciparum 3D7 KRSHHCSVC WCHIKCLCTNPGFLNETFHFEFVSDNTTEY DNNVOMCKKCNLLAI KMDHHCFWIN SCVGLYNOQKYFILLNFVRTKGKYNTNIIKHL FASTA NCBI_PRF_PIR Description line gt DATABANK OF ORIGIN NAME FASTA NCBI_PDB Description line gt PDB NAME CHAT Example gt pd Between The Coagulation Factor X Binding Protein From Snake b 1 KRSHHCSVC OD A Chain A Crystal Structure Of The Complex Venom And The Gla Domain Of Factor X SAK DCSSGWSSYEGHCYKVFKOSKTWADAESFCTKOVNGGHLVS1 HVW GLRAQNKEKOQCS TEWSDGSSISYENW DKCI DKCI TESSGEADFVGOL AQK REESKKCLGVHI E 10 Databanks Formats ETGFHKWENFYCEQQDPFVCEA M FASTA NCBI_PATENT Description line gt pat COUNTRY NUMBER Example gt pat US 4772557VAAHELGXSLGLS FASTA NCBI_GENINFO Description line gt bbs NUMBER FASTA NCBI_GENERAL Description line gt gnl DATABANK OF OR G N DENT Example gt gnl spt 067653 Translation initiation factor MSKLKEYRVNRQIRAKECRLIDENGQOIGIVPIEEALK AEEKGLDLVE IMDYGKFKYELKKKEREARKKOREHOTEVKDIRMKVR IF 3 APOAKPPVCK DEHDLOVKLKHMREFLEEGDKV KVWLRFRGRENITYPELGKKLAERI INELSDIAEVEVQP FASTA NCBI_LOCAL
2. v Container Manager v Expression Analysis v Databank Search v AutoMod Analysis v De Novo Sequencing v BLAST Searching v Data Preparation v Workflow Designer ox Teena 2 Select or clear the check box for each tool to include or exclude the tool in the Tools menu and tool tray 2 4 Setting up ProteinLynx Global SERVER Changing preferences The ProteinLynx Browser Preferences dialog box enables you to change preferences for the search engine processors instrument type bookmarks plate colors and printing To open the ProteinLynx Browser Preferences dialog box either On the toolbar click or Click Options gt Preferences The dialog box has a number of tabs Search Engine see Search Engine tab on page 2 5 enables you to add remove or select a search engine Processors see Processors tab on page 2 8 enables you to add remove or select multiple processors Instrument see Instrument tab on page 2 10 enables you to change the current type of instrument Bookmarks see Bookmarks tab on page 2 11 enables you to specify bookmarks that can be accessed from other parts of the system Colours see Colours tab on page 2 12 enables you to view and edit the plate colors Printing see Printing tab on page 2 16 enables you to specify settings for printing the project or workflow data Search Engine tab Use this tab to add remove or select a search
3. Instrument Spectrum Chromatograr z File Name Instrument 2 1 Theophylinet tab E 2 Theophylline2 7 Inlet Method Eo Theophylline3 3 4 Theophyline4 i 5 phenyltetrazolet MS Method x amp 6 phenyltetrazole2 Icon 5 MS Method 7 phenyltetrazole3 a 8 phenyltetrazole4 5 EN 3 benzimidazole1 10 benzimidazole2 3 MS Tune 11 benzimidazole3 z P N 12 benzimidazole4 2 Inthe MS Method editor click Options gt ProteinLynx Real Time 15 3 Real Time Databank Searching application ProteinLynx Real Time Database Searching i fel Ea File RealTime Settings Help Processing Parameters r Process Method Mass Measure Survey and MSMS MSMS Processing oi yev M r Mass Measure Options r Maxent Lite MV Subtract Min Molecular Mass jo i 5 Peale ie Max Molecular Mass 2000 Databank Searching Below Curve 5 5 a0 Max Charge a 7 F V Smooth Savitsky Golay Threshold an og Window channels 3 Number of Smooths 2 m Peak Centering Min Peak Width atHaf Height i Centroid Top feo Status Processing parameters When the Real Time Databank Searching application is launched the Processing Parameters view is usually displayed If this view is not displayed click the MSMS Processing icon in the tool tray 15 4 Real Time Databank Searching You can change the following parameters Processing parameters Parameter Pro
4. lt REF_ATTRIBUTE NAME SAMPLE_TRACKING_ID VALUE _98375409685408 gt lt REFERENCE gt lt URL PROTOCOL file PATH C temp mass_spectrum_27634 xml gt lt INSERT gt lt UPDATE ELEMENT_TYPE PROJECT gt lt REFERENCE NAME PROJECT gt lt REF_ATTRIBUTE NAME PROJECT_ID VALUE Project3 gt lt REFERENCE gt lt TAG gt lt PROJECT gt lt PROJECT gt lt TAG gt lt UPDATE gt lt QUERY gt C 20 Implementing a plugin for ProteinLynx Global SERVER Query tag definitions in the ProteinLynx DTD Here is the section of the DTD that is specific to plugin activity lt Query Description This describes a query to a plugin and possibly the result of the query Documents Query Attributes username the username for authentication password the password for authentication gt lt ELEMENT QUERY INSERT UPDATE SELECT DELETE TAG gt lt ATTLIST QUERY USERNAME CDATA IMPLIED PASSWORD CDATA IMPLIED lt Insert Description Describes a document to be inserted Document Query Attributes gt lt ELEMENT INSERT REFERENCE URL TAG gt lt Update C 21 Description Describes an element to be updated Document Query Attributes element type the type of element gt lt ELEMENT UPDATE REFERENCE TAG gt lt ATTLIST UPDATE ELEMENT_TYPE CDATA REQUIRED gt
5. Mass Accuracy attributes Attribute Applies to Description Select Calibration MALDI MS The type of calibration that Type MALDI Survey should be performed Electrospray MS INTERNAL should be selected Low Energy when the lock mass is present High Energy in the analyte such as Trypsin autolysis products EXTERNAL should be selected when the data contains dedicated lock mass reference or near point scans External Lock Mass MALDI MS Enter the near point or MALDI Survey external Lock Mass If the Lock Mass is found in the data within the specified tolerance a linear calibration correction will be applied to the data 8 6 Creating custom processing parameters Mass Accuracy attributes Continued Attribute Primary Internal Lock Mass Applies to MALDI MS MALDI Survey Description The primary internal Lock Mass This could be the mass of a trypsin autolysis peptide or another known component of the sample If the Lock Mass is found in the data within the specified tolerance a linear calibration correction will be applied to the data This correction replaces any external correction Secondary Internal Lock Mass MALDI MS MALDI Survey The secondary internal Lock Mass This will be used if the primary internal Lock Mass is not found Lock Mass tolerance All The Lock Mass tolerance If no peak is found within the tolerance no correction will b
6. Result The protein IDs from the selected workflow s are imported into the EMRT result table where appropriate Importing can take some time progress can be monitored in the bottom right corner of the ProteinLynx browser 10 16 Using Expression Analysis to compare and analyze sample groups Searching EMRTs from the EMRT table To search EMRTs 1 Inthe EMRT results table select the Include check box for each cluster you wish to search to select all the clusters see To include exclude all clusters on page 10 13 2 Click the Set Databank Search Parameters button 3 Set the parameters as required see Databank search parameters on page 14 5 for information on the options available 4 Click amp to close the Databank Search parameters window 5 Click the Submit Databank Search button gt 6 Type a title for the workflow and then click OK Result When the search is complete the protein identifications returned are automatically added to the EMRT table for the selected clusters Searching can take some time progress can be monitored in the bottom right corner of the ProteinLynx browser 10 17 Log Plot Viewer To open the Log Plot viewer click Ai To set the values for axes 1 To set the values displayed on the y axis click gt To set the values for the x axis click A 2 Click the values that you want to display on that axis To alter the range displayed on an axis 1 To modify
7. See also If you are unsure how to do this refer to the MassLynx Help Launch the ProteinLynx search engine on the menu bar click Real Time gt Enable Database Search Engine If the program is already running there will be a tick against this menu option Real Time menu Real Time Enable Real Time Processing v Disable Real Time Processing Enable Database Search Engine v Disable Database Search Engine The search engine program accepts processed spectra and identifies proteins which match the spectra If a given number of spectra peptides in other words have matched to a particular protein then the 15 8 Real Time Databank Searching protein is digested and an exclude mass list generated It is possible for the user to set the number of peptides to match a protein before that protein is excluded Rule These database menu items will be unavailable if you have selected remote microkernel see Advanced options on page 15 14 for more details Click Real Time gt Enable Real Time Processing If monitoring is already enabled there is a tick against this menu option Enabling real time processing allows the system to monitor the acquisition system If an acquisition is in progress then the raw data will be processed as it is being acquired Each processed spectrum is then submitted to the search engine for protein identification Set the Processing and Searching Parameters see Processing parameters on
8. Starting modules manually and troubleshooting problems All of the modules are started automatically when you start the ProteinLynx browser on the computer Nevertheless you might wish to start the individual modules separately To start PLGS modules manually 1 Navigate to the PLGS installation directory and then to the bin subdirectory 2 Start the module by double clicking in the GUI or by typing lt module name gt at the command prompt At the command prompt type the following commands SearchEngine to start the search engine PLmicrokernel to start the microkernel ProcessorEngine to start the processor If you start the modules automatically by starting the ProteinLynx browser log files are generated by the software These log files can help you to solve operational problems and will be helpful to Waters if you request technical support To view log files 1 Navigate to the PLGS installation directory and then to the log subdirectory 2 Open the log file in a text editor Two log files are created Processor txt for the processor log SearchEngine txt for the search engine and microkernel log 1 14 Installing ProteinLynx Global SERVER Installing PLGS on UNIX This section describes the steps required to install configure and run PLGS on a non Linux UNIX computer PLGS runs on IBM AIX and Sun Solaris Rule All UNIX commands are case sensitive Before installing PLGS
9. 9 Click File gt Save As Save with the file name Data prep lt current date gt Creating a workflow To create a workflow 1 Click Workflow Designer in the tool tray see Chapter 7 Defining templates for searching with Workflow Designer 2 Click File gt New Select Fragment Ion and then click O 4 Type a title for the workflow Workflow lt date gt for example 5 Right click the workflow node in the workflow frame and then click Add gt Databank Search 6 Set the parameters as shown below Databank Search Query parameters Workflow Designer Databank Search Query Attribute Value Search Engine Type PLGS Databanks SWISSPROT 1 0 Species Peptide Tolerance 100 ppm Fragment Tolerance 0 1 Da Estimated Calibration Error 0 025 Da Molecular Weight Range Oto 200000 Da pl Range Oto 14 Minimum Peptides to Match 1 Maximum Hits to Return 20 Primary Digest Reagent Trypsin Secondary Digest Reagent None Missed Cleavages 1 Fixed Modifications Variable Modifications Exclude Masses Validate Results Yes Filter None Monoisotopic or Average Monoisotopic M alues MH Peptide Charge 2 Instrument Type Default 7 Click File gt Save As Save the workflow as Workflow lt date gt Attaching the data processing parameters To attach the data processing parameters 1 In Container Manager expand the navigator tree so that the Default processing
10. Cone Voltage LockMass Reference Scan Scan Time seconds Frequency seconds Sampling Cone volts Use Instrument Setup Value i Collision Energy volts Mass Measurement Lock Mass A Da Mass Window 1 Da Scans to average E DXC Temperature Correction C DXCON DXC OFF Recommendation When using BEH 75um columns a scan time of 0 6 seconds is suggested 9 Click OK 10 In the method editor click File gt Save As and then save the experiment file with an appropriate name 16 6 Using MSF for qualitative proteomics Running an MS experiment All experiments are carried out through the MassLynx sample list See also For information on configuring and using the sample list refer to the MassLynx Help Necessary sample list fields Only six columns are required within the sample list to carry out an MS acquisition File Name FILE_NAME each raw data file must have a file name File Text FILE_TEXT describes what the sample is MS File MS_FILE the MS Expression method file Inlet File TNLET_FILE the method file for nanoACQUITY Bottle GAMPLE_LOCATION position in autosampler to take sample Inject Volume INJ_VOL amount to inject Tip As column names are configurable they could differ from those given above The field IDs given in brackets above will remain the same whatever the name of the column To add a method file 1 Double click in the M
11. Navigator tree Mass spectrum data not yet obtained Container Manager Microtitre Plates EA Target Plates Default QTOF MSMS At DEBS ERE chimera E Default QTOF MSMS Processing Parameters Templates Workflow Templates Target plate position Raw data spectrum node In this example the instrument QTOF MSMS has been set already See Instrument tab on page 2 10 for information on how to change this 2 Click Set Raw Data File 5 13 5 14 Select Files dialog box for single well Advanced Select Files ma Raw Data Files localhost Sai B ci B Di S B Ui 10 62 2143 Bai N gt B ci B Di gt BE BF gt B o gt S M gt B Ni Attach Processing Parameters Template Choose new template from fi Y Attach Workflow Template Specifty Template vi Specifty Template C PLGS2 2 docs Workflow_ Y vi Simple lt lt Process OK Cancel 3 Select a raw data file from either the local machine or a remote processor Rule You can only select one file 4 Click Advanced to display additional options where you can specify the workflow and processing parameters templates and also process the data 5 Ifyou do not intend to process the data immediately click OK Result The file name is displayed in the Raw Data Spectrum Node Selecting more than one well or spot When setting the raw data it is po
12. To display only unique EMRTs Click the Unique EMRTs Only button i To revert to displaying all the non unique EMRTs click ree To display each identified protein on a separate plot ae Click the Trellis data by protein id button Ea Each identified protein is displayed in its own plot and all unidentified proteins are displayed on one plot To copy the log plot to the clipboard Click the Copy button E The log plot is copied to the Windows clipboard from where it can be pasted into other applications 10 19 Expression Data Viewer Use the Expression Data Viewer to view graphical representations of the relationships between groups samples and replicates You can also view the raw and processed spectra associated with selected replicates To open the Data Viewer click a row in the EMRT or Protein Table and then click k Rule This button is not available if a unique protein is selected in the Protein Table Expression Data Viewer Replicate Levei Rapi ate level data pe ofe Replicate Levei D Processed Spectrum Data Survey Scand jSetection informanion 4 Groupe samet Bem Ferre Gr 5 Saris saieta jihat CO 2 Od 0 Rapar atas soitat Graph Legend Re select Spectra Show an 7 Pras HAADSTOSISPRODAI2S15_041raw Male CG23 a E Practis HAADS72515PRODAI2515_005 raw Green _6 211 FP MOraiar ta HAANTOS IS PANNAGI AWA ras aird OPA Ej
13. Within PLGS is a program called formatdb which produces the index files necessary for BLAST Basic Local Alignment Search Tool searches on a given databank The program requires an environment variable called TMPDIR to be set to a directory with a large amount of free space This directory is used as temporary space by formatdb when it is generating the BLAST indices To display a list of the environment variables use the command set more If TMPDIR is not displayed in the list you need to create it The temporary directory must have read write permissions To create the TMPDIR environment variable 1 Specify a directory that has 1 GB free space or more TMPDIR tmp where tmp is the directory with the free space 2 Toenable large databanks to undergo BLAST formatting without any errors type export TMPDIR Search engine temporary directory The search engine startup script specifies tmp as its default temporary directory This is changed by editing the following entry in the ProteinLynx_SE script Duk co micromass searchenginescratch tmp 1 18 Installing ProteinLynx Global SERVER Change tmp to wherever there is a large amount of temporary space available on the system Typically this could be the same location specified by the TMPDIR variable Running PLGS on UNIX For the AIX version of PLGS there are three components which must be running simultaneously for the system to function These are the search engine
14. Xaas grouping awe Eyes 2 Bk AT 2 ADT reani E22 OMA B D Kiwani D 1 Freel 7 272 Ore There are three levels of view available Group level Sample level and Replicate Spectrum level At each level a number of actions are possible 10 20 Using Expression Analysis to compare and analyze sample groups Control which groups samples or replicates are displayed by selecting or clearing the check boxes below the graph Alter the x axis value by clicking the X Axis grouping value list and then clicking the value you want to use e Select traces or points on the graph by dragging a rectangle over the points you want to select Group level When the Data Viewer is opened it usually appears at Group level If one or more groups are selected the O icon is available Click the icon to go to the Sample level for the selected groups Sample level Click to go back to the Group level If one or more samples are selected click Q to go to the Replicate Spectrum level for the selected samples Replicate Spectrum level Rule For isotopic CAT for example and isobaric GTRAQ for example experiments this level is labeled Spectrum level For other experiment types it is labeled Replicate level In either case the operations available remain the same Click to go back to the Sample level If one or more replicates are selected the Show Processed Data and Show Raw Data D
15. 6 Ifyou want to add other search methods for a Fragment Ion Search the following sequence is suggested 1 Databank Search To identify a set of protein sequences to be analyzed further see Databank Search tool on page 14 3 7 8 Defining templates for searching with Workflow Designer 2 AutoMod Query To characterize the protein sequences fully by considering non specific cleavages amino acid modifications and substitutions see AutoMod Analysis tool on page 14 14 3 De Novo Query To resubmit any fragmentation data that fails to match a peptide see De Novo Sequencing tool on page 14 19 4 BLAST Sequence Homology Query To search novel peptide sequences against a databank to provide matches to homologous proteins see BLAST Searching tool on page 14 238 The attributes and values for each method are displayed in the Editor panel Tip The selected search method is added directly to the node highlighted For example to add an AutoMod Query to a Databank Search Query the Databank Search Query node must be highlighted not the workflow node Typical workflow mi Workflow 22 E Databank Search Query OE G29 AutoMod Query L De Novo Query L BES Sequence Homology Query To reset the template name at any time before saving the template click the workflow node and then click Reset This clears the Title text box and the value of the Title attribute You can then type the new title in the Title
16. Automation Setup dialog box Plugins tab Si Automation Setup Import co micromass plugin FileSystem FileSystemPlugin New Exports JavaPlugin uk co micromass plugin FileSystem Modify Remove Replacing the Import Plugin or adding an Export Plugin You can replace the supplied Import PlugIn but you cannot modify it or add more Import PlugIns However you can modify the supplied Export PlugIn and add new Export PlugIns The dialog boxes are the same for replacing the Import PlugIn and adding Export PlugIns To replace the Import Plugin or add an Export Plugin 1 Click New to replace the Import PlugIn or click Add to add another Export PlugIn You can select from two types of PlugIn Executable or Java Class which have different attributes 2 24 Setting up ProteinLynx Global SERVER Plugin Selector dialog boxes Executable and Java Class Plugin types Plugin Selector Plugin Selector Plugin Type Executable v A Classes Implementing Pluginimp _ Export Selected Results from Container v Save Projects from Browser and PeptideAuto OK Cancel _ Export Selected Results from Container v Save Projects from Browser and PeptideAuto A 2 Add the details to the attribute fields for the Executable or Java Class PlugIn Attributes Executable Plugin Attribute Description PlugIn Name Optional Required on
17. Coverage Pepaiiea Carditence Scare FORTO Seneca sneer Alero Rated TA 1000 F027 Sanan atsam precursor Ag ites 6 roaa 10 Cantaier pom one Rom 3 Oa 2 Ace Arasan Descrgcan Pepttion Contitionce Score BONS Ennan peana Meg Te KICS FOUTOO Sann atann precar Arg AAAS P6 toa 10 7r Cavan yue one fom 4 Oae 1 Ace Aranberria Cavevage Feptiias Caviitence Scare Ce re en TA 100A fT Fodena Gycogenpreupreryeue muci 75297 ab 1000 r75 om w Rw 4 Come f Frasan Descepeany T opite Sore POS onan aeaea ore lenagp I I omens a BY Poad Geicogen piagno muc 73297 zi roaa 1a Cataher pee one Come 2 ce Prosan Descrpean jena Popitas Conlitence Scare a ee 72 1000 1869 PODS Gycogenpreupheryeue muct MMS ab rono 164 Fags 11 5 The toolbar has various functions Print preview toolbar functions Function Description Print Print the project from this screen Import Import another project to be previewed printed or exported Export Export the project results to a csv or html file Refresh Refresh the preview Toggle Preview pages horizontally across the display grid Zoom Increase or decrease the scale of the view range 25 to 200 Use this with the Toggle grid function to display pages across the display as in the graphic Workflow print wizard To use the workflow print wizard 1 You can open a workflow print wizard in two
18. Instrument Type Rule This attribute applies only to Mascot Fragment Ion searches This attribute specifies the instrument that was used to acquire the data which determines the fragment ion series used for Mascot scoring Click the type of instrument in the drop down list 14 13 AutoMod Analysis tool AutoMod increases protein coverage and reduces unmatched MSMS spectra by taking the protein sequences identified through databank searching and rigorously analyzing them against the submitted spectra The analysis can consist of any combination of non specific cleavages post translational modifications and amino acid substitutions The speed of the search is as a consequence of analyzing only those sequences that have already been identified rather than laboriously trailing through the entire databank Tip Using the algorithm in automated workflows see Chapter 7 Defining templates for searching with Workflow Designer can increase coverage and confidence of the top databank search hits while simultaneously filtering out questionable lower scoring hits You can use the AutoMod Analysis tool to search data from any instrument that can generate fragmentation spectra Electrospray Q Tof Maldi PSD and Maldi Q Tof To open the AutoMod Analysis query tool click the AutoMod Analysis Icon QA in the tool tray The AutoMod Search Parameters table opens in the editor panel of the browser 14 14 Query Tools AutoMod Anal
19. OK filter 6 5 10 12 opening Expression experiment 10 3 Expression table 10 10 print templates 11 12 projects 3 5 organizing samples 5 11 P P value filter 10 15 PAM algorithm 14 25 Index 12 matrices 14 25 parameters AutoMod Analysis 14 15 BLAST algorithm 14 24 Consider Modifications 14 16 Consider Substitutions 14 16 Databank Search PLGS 14 4 De Novo Sequencing 14 20 14 21 Expect Threshold 14 25 FASTA Format 13 5 Import Mass Spectrum 5 24 Link from BLAST Results 2 12 MassLynx Directory 2 19 MaxEnt Lite 15 5 Merge Results 5 23 Peak Centering 15 5 PeptideAuto Port 2 19 Process Method 15 5 Smooth 15 5 Subtract 15 5 View Results 5 23 PDQuest files 9 4 XML 3 3 XML file importing gels from 9 6 Peak Centering parameter 15 5 Peak Matching attribute set 8 5 Peak Width 8 13 Peak Width Units attribute 8 16 peaks simplifying 5 26 PepGrab 6 11 PepGrab View 6 11 peptide charge 14 12 data 6 3 sequence 14 25 table 6 15 tolerance 14 6 14 16 view 6 5 6 7 6 9 Peptide Filter attribute 8 11 peptide table 6 13 add remove columns 6 14 change column order 6 15 PeptideAuto Port parameter 2 19 PeptideAuto Server dialog box 5 31 PeptideAuto Server display A 12 peptides specifying modifications 14 21 Perform Deisotoping attribute 8 12 Perform Lock Spray Calibration attribute 8 8 Perform Smoothing attribute 8 10 Periodically Download databank attribute 13 7 Periodically Update databank attribute 13 9 PKL 14 5 format
20. ProteinLynxBrowser To start PLGS using the GUI 1 Navigate to the lt PLGS install location gt bin folder lt PLGS install location gt bin folder File Edit View Go Bookmarks Help d rep 240 A g Back Forward Up Stop Reload Home Location usr local PLGS2 2 bin m p 6 E AutoLynxStub dll blastall exe data fmerge exe formatdb exe 28 0 K 2 8 MB 7 items 137 0 K 4 5 MB gzip exe launcher exe MaldiMonitorServerStu PeptideAuto2 exe PLmicrokernel_AIX64 49 5K 24 0 K b dll 40 0 K 1008 5 K 36 0 K m J PLmicrokernel_Linux PLmicrokernel_ SOLAR Processor ProteinLynxBrowser ProteinLynx_SE 660 7 K IS64 185 bytes 613 bytes 472 bytes 1 1 MB SampleMonitor exe unzip exe 32 0K _ 93 5K 1 12 Installing ProteinLynx Global SERVER 2 Double click the ProteinLynxBrowser file to start PLGS Linux ProteinLynx browser v ProteinLynx Browser StI File View Options Tools B me E BE o Databank Admin Tool p E Databanks s wSsPROT 1 0 Databank Admin Tool Databank Attribute Name Help Type PROTEIN Format FASTA Fasta Format STANDARD_SPACED aaa Location file fusr local PLGS2 dem Make Blastable TRUE Load into Memory TRUE Species for Indexing Management Options Databank Admin Tool Rule The Linux ProteinLynx browser supports the Databank Admin and Help tools only
21. Spota3 on 0001 _ Mass spectrum data not yet ob BE Default MALDI MS 84 3703 135 5937 from test SpotA 2 on 0001 __ Mass spectrum data not yet ob ES Default MALDI MS Processing Parameters Templates S29 Workflow Templates Further icons will be added to represent the plate wells or spots that the samples have been mapped to Importing a gel from an OLB file An OLB file is a system file of a gel image This process only adds a gel image you then have to associate OLB data To import gels from an OLB file 1 In the navigator tree click the Gels node and then right click 2 Click Import Gel 3 Browse to the TIFF tif or JPEG jpg gel image you wish to import Click Open 4 Type the name to associate with the gel in the ProteinLynx browser Result When importing is complete a new node is added to the navigator tree beneath the Gels node Click the new node to display the gel image above the navigator tree 9 5 Gel Manager navigator tree with gel Image Gel Manager Processing Parameters Ternplates Workflow Templates Importing a gel from sample list This process imports a gel image and gel spots To import gels from a sample list 1 Inthe navigator tree click the Gels node and then right click 2 Click Import Gel 3 Inthe Files of Type list click the type of sample list XML file you wish to import PDQuest XML file The sample list XML file that can be exported from PDQues
22. You can zoom in to a specific range along the x axis To view an x axis range 1 Click and drag to select a range along the x axis A red line marks the selected range which is labeled with the maximum and minimum X values in the range and the length of the range The selected range can be adjusted as long as the mouse button is held down Zooming in to a spectrum Precursor mass 471 81 charge 2 e in E 6 RO LOR HF bax Q j 1 l ENI 1 yhtax i i 586 46 i 1 y5 715 53 416 i ih 1 L I 33 j I 1 I I l i 3 176 15 22t y1 i s i 1 1 1 1 t 283 81 446 418 1 162 607 129 01 200 400 600 800 1037 01 M H 2 Release the mouse button The X axis range of the spectrum graph is altered to the selected range 6 24 Viewing results in the Results Browser 3217 07 max Precursor mass 471 81 charge 2 Repeat this procedure as often as needed However the length of the range must be at least 0 001 Da 3 To zoom out again either Right click the Spectrum Viewer once to return to the previous range Right click the Spectrum Viewer twice to return to the initial range the full spectrum Scrolling along the x axis Rule This function is not available when viewing ion probability data To scroll the graph right click and drag along the x axis Displaying a zoomed section of the graph in a separate window Rule This function is
23. lt Select Description Describes a select query Document Query Attributes element type the type of element to return return the type of data to return gt lt ELEMENT SELECT REFERENCE gt lt ATTLIST SELECT ELEMENT_TYPE CDATA REQUIRED RETURN document reference url document gt lt Delete Description Describes a document to be deleted C 22 Implementing a plugin for ProteinLynx Global SERVER Document Query Attributes type the type of element to be deleted gt lt ELEMENT DELETE REFERENCE gt lt ATTLIST DELETE ELEMENT_TYPE CDATA REQUIRED gt lt Tag Description A place holder for any tag Document Query Attributes gt lt ELEMENT TAG ANY gt lt Reference Description This is a reference to an external XML document It is also used to define the element to search for in a query Documents Project Query Attributes name the name of the element gt lt ELEMENT REFERENCE REF_ATTRIBUTE REF_TEXT REFERENCE C 23 gt lt ATTLIST REFERENCE NAME CDATA REQUIRED gt lt Ref_attribute Description This describes an attribute of an element referred to by a reference Documents Query Attributes name the name of the attribute value the value of the attribute gt lt ELEMENT REF_ATTRIBUTE EMPTY gt lt ATTLIST REF_ATTRIBUTE NAME CDATA REQUIRED VALUE C
24. 0689 Glucose 1 phosphate adenylyltransferase large subu 950 3765 0 06876 phospho beta glucosidase EC 3 2 1 86 950 3763 0 0689 Choline O acetyltransferase EC 2 31 6 CHOACTas 950 3763 0 0689 Choline O acetyltransferase EC 2 31 6 CHOACTas 950 3763 0 0689 Choline O acetyltransferase EC 231 6 CHOACTas 950 3763 0 0689 Choline O acetyltransferase EC 2 31 6 CHOACTas 950 3763 0 0689 Hypothetical 39 8 kDa protein Rv2915c 950 3763 0 0689 Kallikrein 11 precursor EC 3 4 21 Hippostasin AMCKCR 1 bMax KCMAHC 1 yMax 951 4471 yMax 10360 023 Counts max 2000 2107 Protein and EST table The top right component of the results browser is a table that displays a list of proteins and ESTs Each row in the table represents a single protein or EST and each column in the table represents a particular data item for example accession number The first column in the table indicates whether the protein match has been set as good OK possible Maybe 0 or poor Not OK Q These assignments are either made manually by clicking in the column to cycle through the options or automatically during searching For details on how and when the assignments are made automatically see Automatic data curation on page B 7 Tip Modifying the assignment for a protein or EST will affect the assignments of its associated peptides 6 12 Viewing results in the Results Browser Pro
25. 105 10 FSS F01023 Apra 2 macroxunenanprecurscr SAAS 100 a ais Canana aom Oae S E 1000 1ST Ace Prose Descrpsnn Popsiion Cantitonce Score food raro WMO Eaa oense Coe a TE mee A002 Apa2 cmacrogeenan orao Got T 1000 laar Container Pom Rar 1 Coin 6 frees Sonartahe ree Aon __ ransn esergann Caverege Poptiisa Carfitanco Soare 1000 1478 FDI0A Commarea POA Cork 65 2 1 1000 tia FOr023 Apra 2 cexcrogeewmnprecurscr SAAI M 1000 a era Come 9 Connie Pow Row t Cone 7 Sonu MEAO pnecuracr 1000 1 dt4 Servet abun precursor 1000 r 203 Fap i Arome yru ReporGenerser Frome yru oporbene Container Pom fom 4 Oae 10 Canana pow one Row 4 Ace Ardan Oain Scare Ace Frasan Descepon average POSES Toran abun precursor Jenang lemaan amg ts n precursor Alp Altos Paoa Senn aban praca sama road 1007 FOO Gecogen pheuRnen Me muck 742385 Coma pm one Row t Comer f dee frase Bescretion _ FOGJAD ADDA DACIANA A POARC ait FSG Apra pmaceusiaue A precursor 716 1000 Cone pam one Rowe t Came f lameng eine Cantinmnce Acc Frans beseripiian FOLIA Apr pancake A precursor FASOD Agta OIII A OOA Contador pom one Ace Prasan Descepean a ABROAD A OOA Alene 602700 Sonus atanan precursor Arp Cavana pom one Ace Protein iencrytion FOUTS Morus auan precursor Amery F272 Senate ponca ACD 1000 Cantans poe one Oae 2 Aee Preset Doscrpaian
26. 16 Threshold Type 8 7 TOF Resolution 8 14 automated task AutoMod Query 7 9 BLAST Query 7 9 Databank Search 7 8 De Novo Query 7 9 automatic data curation 6 12 B 7 Automatic Thresholds attribute 8 13 8 16 Automation Setup dialog box 2 18 2 24 AutoMod Analysis 14 14 14 18 Consider Modifications parameter 14 16 Consider Substitutions parameter 14 16 search parameters 14 16 validate results 14 17 AutoMod Analysis search parameters 14 15 AutoMod Analysis tool 14 1 14 14 AutoMod Query automated task 7 9 filter 7 11 average 14 12 axis assess data quality 10 25 B Backed up folder restoring 1 5 1 11 Background Polynomial attribute 8 10 background subtract type 8 9 Background Subtract Type attribute 8 9 Background Threshold attribute 8 9 backing up PLGS folders in Linux 1 7 PLGS folders in Windows 1 3 BLAST 6 5 BLAST Query 6 7 BLAST View 6 7 make blastable 13 6 13 10 results 6 7 14 26 results panel 14 27 BLAST algorithm search parameters 14 24 BLAST flat file format E 8 BLAST Searching tool 13 6 14 1 14 23 14 27 blastable 13 6 13 10 blocking mode 2 20 BLOSUM algorithm 14 25 matrices 14 25 bookmarks modifying 2 12 removing 2 12 buttons Delete 4 2 7 4 12 6 12 10 13 13 13 14 13 15 Remove 2 8 2 9 2 12 7 4 8 4 13 13 Save 5 22 7 4 8 3 12 5 12 10 13 12 C Calibration File attribute 8 15 calibration type select 8 6 centroid top 8 13 Centroid Top attribute 8 13 change column order 6 15
27. 2 4 toolbars introduction 2 2 preferences button 2 2 Query 14 2 results browser 6 5 Workflow Designer 7 4 tools adding and removing 2 4 AutoMod Analysis 14 14 14 18 BLAST Searching 13 6 14 23 14 27 Container Manager 5 2 Databank Admin 13 2 13 2 13 17 description 13 2 Databank Search 14 3 14 13 De Novo Sequencing 14 19 Digest Reagent tool 12 7 12 10 description 12 7 Expression Analysis 10 2 description 10 2 Gel Manager description 9 2 Modifier tool 12 2 12 6 Print tool 11 2 11 25 description 11 2 Sample Manager 4 2 description 4 2 Troubleshooting installation on UNIX 1 20 Index 18 Linux 1 13 UNIX 1 19 Windows 1 6 Type databank attribute 13 4 U uninstalling PLGS Linux 1 8 UNIX installation troubleshooting 1 20 Update Compression Type databank attribute 13 9 update current project 5 32 A 12 A 24 Update Renew Period databank attribute 13 10 Update URL Address databank attribute 13 9 updating projects 3 5 upregulation 10 11 upregulation filter 10 15 URL Address 13 8 13 9 URL addresses E 2 URL Chooser dialog box 7 10 use replicate filter settings 10 14 user interface 2 2 3 2 V validate results 14 12 14 16 variable modifications 14 11 view column sample lists 5 7 View Results parameter 5 23 viewing 6 34 associated masses 6 34 digest reagents 12 8 exclude masses 6 34 gel image 9 9 gel results 9 9 modifier reagents 12 3 processed mass spectra 5 19 replicates for a clus
28. 2 8 Adhar o protono o a a A 2 8 Modiin Pee onise aia 2 9 Remoyine a PROCEEEDT co idiotccdesmcarcignnieinwee ona mile E TTSS 2 9 Instriie nt ADs ma mune 2 10 PO ae O iiaitie cacao eerie 2 11 Adding a PD okmark sssrin nna teas enss Reratacneie eedeo S 2 11 Modifying a bookmark secrrcnnsnero iire rei E ERRO 2 12 Removing a DOORMATE icine ea 2 12 vi Table of Contents MCAT FU xt ves EEE A AEA EAA AE EN AA AET E A A corte 2 12 Setting confidence levels and colors soossoossosssoososssossroeseroseorrersererrerreeeeee 2 14 Prene e E T 2 16 Setting Automation Setup parameters eessessseessesssssssessssssssssesssosssosssessossso 2 18 Eye ence 81 iena E ne ayers Menne en ree marreren eer eres 2 18 ee CUIG aU EL IALGLLU LA E a mart alate PONE UNA OE Sener oer ere 2 20 PS Ae i EEEE E EE E A ns A A A E 2 23 Replacing the Import PlugIn or adding an Export PlugIn 2 24 Mociiving an Export Plugi o secsssiscssnstsinseossnvesesvecsencsusaewiwneectacrnancerinewets 2 27 Removing an Esport Piel oiar 2 28 3 Creating importing and managing projects e sssssesssssseesseeeccceeeeeose 3 1 Creating a new PTO CCE siroce saonenn iaaea E ENEE SE E EEATT 3 2 Importing and exporting projects e sssesssscesesseeessecceccecsecosoecsscssoccosssscsssscssses 3 3 Opening and updating PLOjects cccccrccsrsessscsssessscsssessscssesoscsssossscossessecesooese 3 5 Dati Peele sis esta eesti ee ee 3 5 Closing and deleting projects cccccs
29. 23 PDQuest 9 4 pkl 2 22 PKL mass spectrum 14 5 XML workflow templates 7 10 XSL 7 11 file permissions changing 1 8 filter Index 8 Expression results 10 13 confidence limit 10 15 P value 10 15 ratio 10 15 replicate 10 14 upregulation 10 15 filters 7 11 14 26 De Novo Query 7 11 for workflow 7 11 print templates curated 11 16 numeric 11 16 text 11 16 XML 7 11 Fine Delta retention time 5 27 fixed modifications 14 10 14 16 format FASTA 14 18 significant clusters file 10 24 Format databank attribute 13 4 fragment ion display 6 20 tolerance 14 7 14 16 14 21 fragment data low and high energy 7 5 Fragment Intensity Threshold attribute 8 15 Fragment Matching Window attribute 8 15 Fragments attribute 12 5 G gapped 14 26 gel adding 9 3 image 9 6 location of gel spots 9 9 manipulating 9 9 showing axis labels 9 9 viewing 9 9 zooming 9 9 importing 9 3 importing from PDQuest XML file 9 6 importing from Progenesis XML file 9 6 results viewing 9 9 spots adding without image 9 3 circled 9 9 importing 9 3 location on gel image 9 9 Gel Manager 4 5 9 2 9 10 description 9 2 processing data 9 8 replacing a sample 9 7 Genbank flat file format E 6 generating processed samples 4 5 glu fibrinopeptide B A 14 Graphical Data 11 14 H high energy fragment data 7 5 homology threshold B 7 host 2 20 hyperlinks databanks 4 4 ICAT experiments 10 21 icons AutoMod Analysis 14 14 BLAST Searching 14 23 Container
30. AT1G69120 68300 M06877 F4N2 9 HOMEOTIC PROTEIN BOI1lAP1 PUTATIVE SIMILAR TO HOMEOTIC PROTEIN BOI1AP1 GI 1561777 FROM BRASSICA OLERACEA SUPPORTED BY FULL LENGTH CDNA CERES 39890 ATGGGAAGGGGTAGGGTT CAAT TGAAGAGGATAGAGAACAAGAT CAATAGACAAGTGACA TTCTCGAAAAGAAGAGCTGGTCTTTTGAAGAAAGCTCATG AGATCTCTGTTCTCTGTGATGCTGAAGTTGCTCTTGTTGTCTTCTCCCATAAGGGAAAAC TCTTCGAATACTCCACTGATTCTTGTATGGAGAAGATACT TGAACGCTATGAGAGGTACT CT TACGCCGAAAGACAGCT TAT TGCACCTGAGTCCGACGT CAATACAAACTGGTCGATGGAGTATAACAGGCTTAAGGCT AAGATTGAGCTT TTGGAGAGAAACCAGAGGCATTATCTTGGGGAAGACTTGCAAGCAATG AGCCCTAAAGAGCTTCAGAATCTGGAGCAGCAGCTTGACA CTGCTCTTAAGCACATCCGCACTAGAAAAAACCAACTTATGTACGAGT CCATCAATGAGC TCCAAAAAAAGGAGAAGGCCATACAGGAGCAAAACAGCAT GCTTTCTAAACAGATCAAGGAGAGGGAAAAAATTCTTAGGGC TCAACAGGAGCAGTGGGA TCAGCAGAACCAAGGCCACAATATGCCTCCCCCTCTGCCA CCGCAGCAGCACCAAATCCAGCATCCTTACATGCTCTCTCATCAGCCATCTCCTTTTCTC AACATGGGTGGTCTGTATCAAGAAGATGATCCTATGGCAA TGAGGAGGAATGATCTCGAACTGACTCTTGAACCCGTTTACAACTGCAACCTTGGCTGCT TCGCCGCATGA FASTA NRDB NRDB is the same subtype as NCBI_LEXPASY_STANDARD FASTA UNIGENE Description line gt gnl UG UGAccession DESCRIPTION gb gi ug len Example gt 2386477 gnl UG Hs S2386477 PM3 FT0024 240500 001 f10 Homo sapiens cDNA gb BE769099 gi 10222757 ug Hs 1287 len 384 CTCTGAGATCCCCACTTCCAGAGTAGTATAAGATGTTATCCGCCCTCCAGGAGCTTACAA AAC TAGAGGCAGAAATAAGATGTACATGTGACTCAGGCAGCATGTGACACACA
31. AVERAGE sernir onara NE 14 12 IE Rh Tc aea Seven rennin re eae e MeMR Seo celle UNS rer een OPIS 14 12 Wage Cae Lom harot esir 14 12 SEP UINONE TVS cossiro aran EA 14 13 AutoMod Analysis tool ssssiissicsieceeasesssissdsissesiisdsissdeiasssssssesaiaseiavsesaasesssassaeiaees 14 14 AutoMod Analysis search parameters cccccccccccccccccccecceecccecceeceseceecceeeeseees 14 16 Consider Modifications ica cr crepe hci cians ors ented tenia 14 16 Consider Sabstitutions suorina iai EAE A RE 14 16 Specifying the maximum substitutions and modifications per peptide 14 16 Specifying the likelihood of substitutions ccccccccccccccccceceeceeeeeeeeeeeeee 14 17 Validate RESUS cornia E E ene eee 14 17 Selecting protein sequences for the search ccccceccccccccceeceeeceeeceeeeeeees 14 18 Selecting EST sequences for the search ccccccccccccccceceeceeceecceeeeeeceeeees 14 18 De Novo Sequencing tool sssssssssssssssesssnsssssnssssscssssssssesscsesscssccssccsscsscsess 14 19 De Novo Sequencing paraMeters ccceccccccccceccscsesssccecceeeeecusaeenesesceseeeeeaaes 14 21 Specifying the estimated calibration error ccccceecceeesscceeeeeeeeeeeraaees 14 21 Specifying maximum hits to POW sisi cc discccasisiearseisisoiaaisicarsdiaaseiisieeins 14 21 Specifying modifications to Peptides cccsesscccecceeeesessneeeeeeeeeseenaaas 14 21 Vahdate Resit seisi a 14 22 BLAST Search itig tool sisccesevseseocsczesescscecedsevedesecswsdsaues
32. Also at a user defined time a reference scan is sampled from the NanoLockSpray reference sprayer 16 2 Using MS for qualitative proteomics Creating an MS method file The low and elevated collision energies are set up from within the MS Method editor in MassLynx The ideal values to set for an experiment can vary depending on your hardware setup The values shown in the screen shots that follow are suggested when using Atlantis 75um or 300um columns with a nanoACQUITY UPLC Suggested values when using a BEH 75um column are also given In all circumstances some experimentation might be necessary to find the optimal values for your requirements To create an MS experiment file 1 Inthe MassLynx shortcut bar click MS Method MS Method editor Experiment Setup c masslynx myexpression pro acqudb default exp File Edit Options Toolbars Functions Help Ci S ZX v E Msscean P mee E Survey Total Run Time 0 00 e No Type Information y O 2 Delete the default function that is present in the function list 3 Click ZEmeson to open the Expression function editor 4 On the Acquisition tab enter the values as shown Tip The Start and End times mirror the LC gradient The times shown below relate to a 90 minute gradient 16 3 Acquisition tab Acquisition TOF MS Expression Cone Voltage LockMass r Acquisition Times Total time for this acquisition Start Time B mmtes End Time fo o o m
33. Description line DENT F ER gt lcl Example gt lcl 067653 Translation initiation fac KKEGNFM I tor IF 3 MSKLKEYRVNRQIRAKECRLIDENGQQIGIVPIEEALK AEEKGLDLVE IMDYGKFKYELKKKEREARKKOREHOTEVKDIRMKVR KVWLRFRGRENT YPELGKKLAERI INELSDIAEVEVOQP FVLAPKRKK APOAKPPVCK DEHDLOVKLKHMREFLEEGDKV KKEGNFM I FVLAPKRK FASTA PDB Description line gt NAM E CHA N D ESCR PT ON Example gt 1C8F A FELINE PANL EUKOP EN A V VVE G GVGISTGTFNNOTEFKFLENG ALDDIHVEIVTPWSLVDANAWGV ATOPPTKVYNNDLTASLMVAL TLIPSHTGTSGTPTNVYHGTDPD HTWOTNRALGLPPFLNSLPQSEGATNFGDI AAG GYSAPYYSFEASTOGPFRKTPI RUS CAPSI TANSSRLVHLN DVOFYT RGGAQTD RF TY TAHODTGRYPEGDWIOQN LNNVPPVYPNGOQIWDK NMSRIVTYS QLAPRKLY FASTA PIR Description line gt ACC RI ESS ON P release Example NFNLPVTN EFDTDLKPRLH DFWWKGKLVFKAKLRASHTWNP VENPGDWOLI DSNNTMPFTPAAMRSETLGFYPWKPTIPT HLLRTGDEFATGTFFF RGVTOMGNTDY DGDPRYAFGROHGORKTTTTGETPE DPIGGKTG INAPFVCONNCPGOQLFVKVAPNLTNOYDP ENSVPV GVQQDKRI ENQAA DNVLLPT G MPE MSEL VNT QQMS REL EASE NUM gt 952288 PIR2 releas MPSKKVLOTEHIN
34. Estimated Calibration Error Da or ppm on page 14 7 This increases the quality of the validated peptide returned To specify the tolerance type the value into the text field Estimated Calibration Error Da or ppm Restriction This attribute is not available for Mascot database searches The Estimated Calibration Error is an estimation of the error introduced following instrument calibration This value is fundamental to the scoring of a peptide sequence against a given fragmentation spectrum As a tight error will significantly reward well measured data in the scoring it is recommended that spectra submitted are well mass measured to allow a low Estimated Calibration Error to be set It is not necessary to adjust the estimated calibration error for small variations of this number in the fourth decimal place When comparing calculated peptide or fragment masses with the data it is important to know how well the masses in the data are determined If this estimate is good the information that can be extracted from the data is maximized A good estimate will increase the scores of correct identifications 14 7 Suitable values differ between instruments Recommended values are Estimated Calibration Error recommended values Estimated Calibration Error Instrument Detail recommended value Equipped with nano lockspray 20 ppm MALDI equipped with internal 30 ppm lockmass MALDI equipped with external 5
35. ID Elements for deletion are selected in the same way as in a select query The only difference is that the query action is a DELETE rather than a SELECT Note that there is no return type as no document can be returned after it has been deleted The above example has selected the Mass Spectrum document for Sample Tracking ID B001 to be deleted Implementing a plugin for ProteinLynx Global SERVER Insertion of documents lt xml version 1 0 gt lt QUERY gt lt INSERT gt lt TAG gt lt WORKFLOW gt lt WORKFLOW gt lt TAG gt lt INSERT gt lt UPDATE ELEMENT_TYPE PROJECT gt lt REFERENCE NAME PROJECT gt lt REF_ATTRIBUTE NAME PROJECT_ID VALUE Project3 gt lt REFERENCE gt lt TAG gt lt PROJECT gt lt PROJECT gt lt TAG gt lt UPDATE gt lt QUERY gt Inserting a Workflow document and updating the associated Project document Documents can be inserted either by specifying the entire document or by specifying a URL at which the documents can be found In the above example a workflow is to be inserted The entire workflow document is located in the INSERT block and this is then followed by an update query for the Project with the PROJECT_ID Project 3 Alternatively a URL can be provided inside a REFRENCE element as illustrated in the following example code lt xml version 1 0 gt lt QUERY gt lt INSERT gt lt REFERENCE NAME MASS_SPECTRUM gt
36. If the mass spectrum data contains peak retention times as well as masses you can choose to display either retention times or masses on the x axis of the Spectrum Viewer To change the x axis view click T to show retention times on the x axis or T to show masses on the x axis If retention times are displayed along the x axis the most intense peaks will be annotated with the peak mass If masses are displayed along the x axis the most intense peaks will be annotated with the peak retention time or with the peak mass if the spectrum data does not include retention times Viewing the fragment ion display To view the fragment ion display click the button to the right of the spectrum view 6 20 Viewing results in the Results Browser The fragment ion display shows the expected masses of the fragment ions for the predicted peptide sequence and the related delta masses of the experimental value Ions that are shown in gray are undetected ions in the spectrum and therefore do not have corresponding delta masses The ions found are colored according to the type of ion using the color scheme on the MSMS spectrum display Gray The peak is not matched to a peptide from the current protein or EST Blue A standard peptide that is with no modifications or missed cleavage sites Red A peptide that contains one or more missed cleavage sites Green A peptide that contains one or more post translational m
37. Importing this file type imports any project and gel information specified in the XML XML file The ProteinLynx Global SERVER project XML file Using this import option allows you to explicitly specify project and project member ids The XML is validated against the Protein Lynx Global Server XML schema Caution This option will not import data or results It should only be used to import a skeleton project that includes sample and container information ZIP file A ProteinLynx Global SERVER zipped project created by exporting a project from PLGS Click Open Result The project is imported into PLGS and then opened Depending on the size of the project imported the process can take some time The status bar in the bottom right of the browser indicates that the import is in progress To export a project 1 2 Click File gt Export Project Navigate to the directory in which you want to save the exported project and type a name for the file Click Save 3 3 Result The project is exported as a compressed zip file which can then be imported into another PLGS installation 3 4 Creating importing and managing projects Opening and updating projects To open a project 1 Inthe tool tray click the icon for one of the tools that requires a project Sample Manager Gel Manager Container Manager or Expression Analysis 2 Click the Projects box in the PLGS toolbar to display the projects list
38. MS Low Energy High Energy The number of consecutive Lock Spray spectra which should be summed to determine the mass correction for each precursor Creating custom processing parameters Noise Reduction attributes Not all attributes are available for all panels check the Applies to column in the table below to see whether the attribute listed relates to the panel you are configuring Noise Reduction attributes Attribute Background Subtract Type Applies to All Description Background subtraction removes slowly varying low frequency components from the data This can improve the results of subsequent processing Select from None No background subtraction is done Normal Normal background subtract removes smooth slowly varying components from the data Adaptive Adaptive background subtraction additionally removes noise with a structure that repeats every nominal mass roughly 1Da Adaptive background subtraction can be particularly useful for low concentration MALDI data Background Threshold All The algorithm will aim to find a smooth function which lies above this percentage of data points The value of the function in each channel is then subtracted from the data 8 9 Noise Reduction attributes Continued Attribute Background Polynomial Applies to All Description The order of the polynomial with which to fit the bac
39. Manager 5 2 Data Preparation tool 8 2 Databank Search 14 3 Databank Searching 15 5 Digest Reagent 12 7 real time status 15 7 sample list view column 5 7 spectrum 5 18 workflow 5 18 WorkFlow Designer 7 2 identity threshold B 7 Import Gel Spots dialog box 9 3 Import Mass Spectrum parameter 5 24 Import PlugIns 2 23 Import Worksheet command 5 30 A 10 A 20 importing gel 9 3 gel spots 9 3 mass spectra 5 22 projects 3 3 significant clusters 10 24 include clusters 10 13 index for PepGrab 13 6 Index For PepGrab databank attribute 13 6 influence 6 23 Installation troubleshooting on UNIX 1 20 installing in a client server environment 1 1 on AIX 1 15 on Linux 1 7 on Windows 1 3 services 1 4 instrument specifications A 1 B 1 type 14 13 Intensity Range attribute 8 11 Index 9 Intensity Threshold attribute 8 7 interfacing with MassLynx 5 29 internal standards 10 9 10 10 ion display fragment 6 20 probabilities 6 22 IP address 1 4 2 6 2 20 isobaric experiments 10 21 isotope labeled samples 10 5 iterations 8 12 Iterations attribute 8 12 iTRAQ experiments 10 21 K Keep Archives databank attribute 13 10 L label free analysis 10 5 Link from BLAST Results parameter 2 12 Linux installation 1 7 Load into Memory databank attribute 13 6 Location databank attribute 13 6 lock mass external 8 6 lockspray 8 8 primary internal 8 7 secondary internal 8 7 tolerance 8 7 Lock Mass tolerance attribute 8 7 Lock Spra
40. Modify the details as required 4 Click OK Removing a bookmark To remove a bookmark click a bookmark and then click Remove Colours tab Use the Colours tab to view and edit the well or spot colors that are shown in the target plate graphic in the Container Manager display see Creating a new vial microtitre or target plate on page 5 9 The colors show the status of a microtitre plate well or target plate spot and when appropriate the confidence level of the top scoring hit 2 12 Setting up ProteinLynx Global SERVER Preferences dialog box Colours tab Preferences Confidence Level 95 or above 50 10 0 1 Less than 0 1 No results No data Selected well or spot The confidence levels and colors shown are the defaults Default plate color descriptions Well or Spot State Confidence Level Color High score 95 or above Green Medium score 50 Yellow Medium low score 10 Light orange Low score 0 1 Orange Very low score Less than 0 1 Red No results Blue No data Gray Selected well or spot Black 2 13 Setting confidence levels and colors You can adjust the confidence levels of results that trigger the display of the colors in the wells or spots To set the confidence levels and colors 1 Use the slider bars to adjust confidence levels 2 To change a color associated with a confidence level click the color The Select a Colour dialo
41. OLB file A Waters format OLB file that maps samples from the gel onto plates 9 3 Import Gel Spots dialog box parameters Continued Parameter Description PDQuest export A PDQuest export file listing the co ordinates of file spots that were excised from the gel to create samples PDQuest files must be in plain text txt or excel xls format OLB files must be in the Waters olb format olb 3 Select the Plate Type 4 Use the Browse buttons to select the relevant OLB and PDQuest files Rule Both the OLB file and the export file must be specified 5 Click OK The Specify Plates dialog box opens Specify Plates dialog box Waspecify Plates Eg Plate 1 Create new plate record Barcode Update existing plate record ox cance 6 Select a plate from the ProteinLynx system or create a new plate record Rule If a new plate is created a title or identifying number must be entered If there is more than one plate listed in the OLB file then there will be a prompt for each plate Results The specified plates are produced or updated as necessary in the Container Manager e When importing is complete nodes are added beneath the gel node in the navigator tree to represent the imported gel spots 9 4 Viewing and processing gel data with Gel Manager Gel Manager navigator tree gel data imported testi amp cels a m 49 95 34 4424 from test
42. PlugIn Selector dialog boxes Executable and Java Class PlugIn types on page 2 25 which contains the details of the PlugIn 3 Modify the details as required and then click OK 4 On the PlugIns tab click OK Requirement For the PlugIn changes to take effect the ProteinLynx browser must be restarted gt Removing an Export Plugin Rule You can only remove an Export PlugIn when there is more than one in the list To remove an Export Plugin 1 In the PlugIns page select the PlugIn from the list and then click Remove The PlugIn is removed from the list 2 Click OK Requirement For the PlugIn changes to take effect the ProteinLynx Browser must be restarted 2 28 Setting up ProteinLynx Global SERVER Creating importing and managing projects You organize your work in ProteinLynx Global SERVER using projects Each project contains a collection of related settings files and data that represent an area of work Many of the tools you work with in PLGS create and manage settings and templates that can be applied across projects These tools do not require a project to be created or opened Sample Manager Gel Manager Container Manager and Expression Analysis require that a project is created or opened before they can be used Contents Topic Page Creating a new project 3 2 Importing and exporting projects 3 3 Opening and updating projects 3 5 Closing and deleting projects 3 6 3 1 Creating a n
43. Result The MassLynx sample list will be updated Acquiring data Once the sample list is imported into MassLynx data can be acquired in the normal way Running the sample list opens the PeptideAuto Server dialog box which monitors the acquisition To acquire data 1 Inthe main MassLynx window click gt to open the Start Sample List Run dialog box 2 Select Acquire Sample Data and Auto Process Samples 3 Click OK The PeptideAuto Server dialog box is opened which monitors the progress of the acquisition MassLynx starts to acquire and process data 5 31 PeptideAuto Server dialog box MassLynx YaPeptideauto Server ojx Messages 22 01 03 14 13 32 STATUS In FileSystemPlugIn process 22 01 03 14 13 32 STATUS Query has been filtered 22 01 03 14 13 32 INFORMATION Searching for Project xml 22 01 03 14 13 32 INFORMATION Getting the sampleTracking element from the project xml 22 01 03 14 13 32 INFORMATION Getting the processing parameters template ref 22 01 03 14 13 32 INFORMATION Got the processing parameters template ref 22 01 03 14 13 32 INFORMATION Getting the processing parameters template element 22 01 03 14 13 33 INFORMATION Got the processing parameters template element 22 01 03 14 13 33 INFORMATION Deriving the search type from the instrument mode 22 01 03 14 13 33 INFORMATION Got the spectrum data to send to the processor 22 01 03 14 13 33 INFORMATION Getting the workflow template ref
44. Spray Calibration Yes Lock Spray Lock Mass 716 4585 Dale Lock Spray Scans 2 Lock Mass tolerance 0 1 Da Perform Lock Spray Calibration Enable or disable Lock Spray calibration Enable for data acquired using an external Lock Spray interface Perform Lock Spray Calibration v A 15 7 Set the Noise Reduction attributes in the Electrospray Survey and MSMS panels as shown below Noise Reduction attributes Electrospray Survey panel Data Preparation Electrospray Survey Background Threshold Background Polynomial Perform Smoothing Smoothing Type Smoothing Iterations Smoothing Window Noise Reduction attributes MSMS panel Data Preparation MSMS Attribute Value Background Subtract Type Normal Background Threshold Background Polynomial Perform Smoothing Smoothing Type Savitzky Golay 3 channels 8 Set the Deisotoping and Centroiding attributes in the Electrospray Survey and MSMS panels as shown below A 16 Quick Start Tutorials Deisotoping and Centroiding attributes Electrospray Survey panel Data Preparation Electrospray Survey Value Perform Deisotoping Yes Deisotoping Type i Iterations Automatic Thresholds Threshold Minimum Peak Width Centroid Top TOF Resolution Perform Deisotoping Deisotoping Type Automatic Thresholds Threshold Minimum Peak Width TOF Resolution 10000 NP Multiplier 0 7
45. Successful 1 5 Modify Bookmark 2 12 Modify Processor 2 9 Modify Search Engine 2 7 Index 6 New Container Tool 5 9 PeptideAuto Server 5 31 PlugIn Selector 2 25 ProteinLynx Browser Automation Setup 2 18 ProteinLynx Browser Preferences 2 5 5 33 Select a Colour 2 14 2 15 Select Files 5 15 single 5 14 Select Processing Parameters A 9 A 19 Specify Plates 9 4 Start Sample List Run 5 31 A 11 A 21 URL Chooser 7 10 digest fragments Protein Workpad 6 30 Digest Reagent tool 12 7 12 10 description 12 7 digest reagents adding 12 9 deleting 12 10 editing 12 9 non specific 14 10 saving 12 10 viewing 12 8 displaying columns 6 14 ion probabilities 6 22 real time diagnostics 15 15 displays PeptideAuto Server A 12 docs folder 1 5 1 11 Download Compression Type databank attribute 13 8 Download Renew Period databank attribute 13 8 Download URL Address databank attribute 13 8 downregulation 10 11 dta format 2 22 dynamic bookmark 2 11 E edit precision peptide or protein table 6 14 editing databanks 13 11 digest reagents 12 9 modifier reagents 12 4 workflow templates 7 9 Electrospray DDA 8 5 Electrospray High Low 8 5 Electrospray MS 7 2 7 5 8 5 Electrospray Shotgun 7 2 7 5 EMBL E 3 EMRT table 10 9 export switch lists 10 23 import significant clusters 10 24 view replicates for cluster 10 12 viewing 10 10 End Time 13 10 enzyme 14 9 Error Messages 6 19 erythromycin A 14 EST data 6 3 table 6 12 EST sequences s
46. and printing project data 1 2 Managing modifier and digest reagents Use the Modifier and Digest Reagent tools to manage the modifier and digest reagents used in the system Contents Topic Page Getting Started with the Modifier tool 12 2 Viewing existing modifier reagents 12 3 Adding and editing custom modifier reagents 12 4 Getting started with the Digest Reagent tool 12 7 Viewing existing digest reagents 12 8 Custom digest reagents 12 9 12 1 Getting Started with the Modifier tool The Modifier tool enables you to manage all modifier reagents used in the ProteinLynx system With it you can perform these tasks View the properties of the large number of modifier reagents that are supplied with ProteinLynx Define your own modifier reagents which are immediately available to the full suite of ProteinLynx browser tools gt S To open the Modifier Tool click the Modifier Tool icon on the tool tray A list of modifier reagents is displayed Supplied reagents are shown in gray text custom user defined reagents are shown in black text Any modifier whether supplied or custom can be used in an isotopically labeled experiment so long as its Quantitation Reagent attribute is set to Isotopic 12 2 Managing modifier and digest reagents Viewing existing modifier reagents To view the properties of a reagent click a reagent in the list The attributes and values are displayed in the panel below the l
47. any databank that resides on the local computer To remove the databank from the machine but not remove the files associated with the databank 1 In the navigator tree click the databank to be removed 2 Click the Remove button t on the toolbar 3 Confirm the request when prompted Results The databank is removed from the record of the Databank Admin Tool The databank will no longer appear in the navigator tree and will not be available for searching by the various ProteinLynx tools The files associated to the databank including the flat file of sequences will not be removed from the computer Deleting databanks Using the Databank Admin Tool you can delete any databank that resides on the local computer To delete a databank from the machine including the files associated with the databank 1 In the navigator tree click the databank to be deleted 2 Click the Delete button on the toolbar 13 13 3 Confirm the request when prompted Results The databank is removed from the record The files associated to the databank including the flat file of sequences are deleted from the computer Any auxiliary files used for BLAST searching and any archive files are also deleted e The databank no longer appears in the navigator tree and is not available for searching Deleting archive files You can delete the archives of any databanks that reside on the local computer To delete an archiv
48. data on page 5 13 the samples can be exported to MassLynx as a sample list Requirement Some familiarity with MassLynx is needed Refer to the MassLynx Online Help for details To export a sample list 1 Right click the plate or vial node and then click Export Sample List to MassLynx 5 29 Export to MassLynx dialog box zi Export to MassLynx Project MALDITestProcedure PRO amp Method File malditest exp v Inlet File Default Tune File None v FileName MALDI Test Listt Data Name Sample_ More gt gt Samples included in a previous list will not be included in this list nless data acquisition was cancelled before spectra were obtained e default injection volumes will be used for the exported samples inless the olb file is imported into MassLynx and the injection volume or each sample edited manually 2 Select A Project to export to An MS Method file from the drop down list An appropriate Inlet file for Q Tof MSMS only A Suitable Tune file A File Name for the MassLynx sample list An MS Data Name Click Export Open MassLynx Click File gt Open Project to open the relevant project D gU a apo Click File gt Import WorkSheet to import the olb file Navigate to the relevant MassLynx project and click the olb file with the name you specified 5 30 Specifying samples vials and plates with Container Manager 7 Click Open
49. details on starting the processor see Chapter 1 Installing ProteinLynx Global SERVER The raw data file requested was not found The raw data viewer needs the original raw data file to be present Ensure that the raw data file has not been deleted or moved since processing Invalid spectrum format please re process data This indicates the spectrum is in an old format Process the raw data again to update the spectrum The data requested was unavailable please try again This means the processor is running out of memory and has cleared the data Try to reselect the node in the tree if that does not work restart the processor Results browser 6 19 Viewing raw data error messages Continued Error Message The processor experienced an internal error Please examine processor output Suggested Course of Action This is an internal error and should be reported to Waters Attach either the log file see Chapter 1 Installing ProteinLynx Global SERVER for assistance on locating the file or a screenshot of the processor window Ctrl Print Sern to an e mail and send it to your local Waters support representative The processor did not accept the request This is an internal error and should be reported to Waters Request parameters were invalid no data was available This is an internal error and should be reported to Waters Changing the x axis view
50. flavour gt bin lt destination gt Example cp PLGS2 2 5 SOLARIS SPARC bin usr local Use the chmod command to set up permissions on the installer package so that it can be executed chmod 777 PLGS2 2 5 lt unix flavour gt bin Once the permissions have been set the installer package is ready to be executed Type the following command in the directory that the package is in to execute the installer package PLGS2 2 5 SOLARIS SPARC bin or PLGS2 2 5 AIX bin The installer user interface can take a while to appear The first welcome screen advises you to ensure that you have uninstalled any previous versions Tip Occasionally the installer user interface can appear blank If this occurs close down the installer and restart it with the command in step 5 Read and understand the terms of the license agreement Click Accept in the License Agreement screen and then click Next The Destination screen opens Rule You cannot install PLGS in a directory that has spaces in the name If you attempt to do so you will be prompted to enter the path again 1 16 Installing ProteinLynx Global SERVER 10 In the text field specify a new or empty directory in which to install the program the directory should not contain any previous PLGS files If the directory does not exist the installer creates the directory automatically Confirm that your installation details are correct A progress indicator on a splash screen shows the
51. from masses found in the data Therefore due to the possibility of misassignment a detected mass being mistaken for a different theoretical mass the corresponding data is suppressed according to how well the masses match the theoretical masses rather than being completely extinguished Copying data To copy the data in a table to the clipboard right click either the protein or peptide table and then click Copy Table Data The data copied to the clipboard is organized by row Each line of copied text represents a single row the line lists the row number and the data values from the table Separate data values are comma separated Printing the results To print a summary of the workflow results right click either the protein or peptide table and then click Print Workflow Printing is controlled using the Print wizard see Using print wizards on page 11 3 Spectrum Viewer for MS data For a search with an MS spectrum the bottom component of the results browser is a graphical display of the MS spectrum data used for the search For a search with an MSMS spectrum the middle component of the results browser is a graphical display of the parent spectrum from the MSMS spectrum data used for the search 6 16 Viewing results in the Results Browser Spectrum Viewer for MS data ma x 29429 67 100 822 1000 1276 773 1500 2000 M H In the graph X axis retention time Rule X axis mass if
52. graphic To add horizontal rules 1 Right click the Header node and then select Insert gt Horizontal Rule 11 21 Print Tool adding horizontal rules Ya Protein ynx Browser Print Tool SampleProjectTemplate Proteintynx Gloda SERVER V2 2 5 Report Generator D HeaderFooter I Sample Manager Gel Manager Container Manager 8 Expression Analysis Header j E Paragraph H L Footer LE Field Introduction C Template Details L E image D Results C Project content Locations content J Hits content C Proteins content L C Peptides conte Horizontal Rule Element L summary 5 6 S ee ee e 6 aloe Databank Search AutoMod Anat 3 De z Sequencing BLAST Searching Element Properties Data Preparation on A amp Print Tool 2 Use the settings in the tabs to change the line style and dimensions of the rule To add fields 1 Right click the Footer node and then click Insert gt Field 2 In the page click the Field box to open a drop down list 3 In the list click Page Number 11 22 Creating print templates and printing project data Print Tool adding fields File Edit Insert View Options Tools Ban 68 2 amp amp oi Sample Manager Gel Manag
53. icons become available To display raw or processed data 1 In the Replicate level graph select traces or points by dragging a rectangle over the points you want to select 10 21 2 Click to display processed data or to display raw data 3 Select the check boxes beside the replicates you wish to view spectra for 4 Click Show Selected Result The selected spectra are displayed on a single graph To show or hide the spectra on the graphical display select or clear the check boxes in the Graph Legend section To select different replicates for display click Re select Spectra and then repeat steps 3 and 4 above To switch back to the data profile view click i Tip Switching to the profile view does not reset your spectra selections Click the appropriate icon to revert to the spectra view 10 22 Using Expression Analysis to compare and analyze sample groups Exporting Switch Lists Clusters can be exported from EMRT results tables as switch lists To export clusters as a switch list 1 Inthe EMRT table see EMRT table on page 10 10 select the check box in the Include column beside each cluster you wish to include See To include exclude all clusters on page 10 13 for details on including all clusters 2 Click Export Switch List z 3 Inthe Export Switch List dialog box browse to the location you wish to save the file in and then type a name for the switch list file 4 Click Save Result
54. ion probabilities nerusssoniorisitii kari ana 6 22 Spectrum Viewer options cccccccccccccccccccccceccceceeccceccssccceccesceseccseceseessvenss 6 24 CODAE a ores crta aceasta aaa 6 26 Protein Workpad sssssssavzsssesstedcnevsdsicicesbeckacacs haces ee Rao ek hahaa 6 27 OVO A ee THAD E E E A A E E T E eae 6 28 Running a simulated digest c cii lt ssevsssseusvessavevseevensateneaversvevenverentaverenasvess 6 29 Retrievinie databank Gntries ccccccsssccincccivncousmesweeimetancaisansvawenareniwness 6 30 Exclude Masses Work pa cccsccssssssessssssssssessscssssssssssssssssssssssssssssssssssnseses 6 31 Adding items to the excluded list s sssseseseesseseseeeeeseecessnteeeeeeanenes 6 32 Deleting items from the excluded list cccccccccccsceecncesccecceceeeteeeceeceeess 6 33 Running a simulated digest for a protein c cccceeccecceeseeceeceeeeeeteeeeeeeeeess 6 33 Viewing the masses associated with an excluded item 0eceeee 6 34 Table of Contents ix 7 Defining templates for searching with Workflow Designer 7 1 What is Workflow Designer siisiisiscssisiesssssaesasescdcsssesssancdiasccstacesasseasassaianesaanaes 7 2 The Worktlow Designer miter ee ocornseneian ennea EA 7 2 Workilow Designer toolbar iisicciiscssiasssssnissincseionsesierieterssuawinesnevensveeessveouaveseanie 7 4 Creating a workflow template sccsssccsscasesesactcxesisaseessensssasasnesesarecsestagnstasnanmeccase 7 5 Editing wor
55. lt residues or terminus gt Name 12 4 Managing modifier and digest reagents Reagent attributes Continued Attribute Modifier type Description A modifier applies to one of three sites of a protein the SIDECHAIN N TERM or C TERM Choose one from the drop down list If a modifier can apply to both sidechain residues and termini define a different reagent for each case Quantitation Reagent Whether this reagent should be considered a quantitation labeling reagent to be used in isotopic ICAT for example or isobaric i TRAQ for example labeling experiments Rule To be considered the reagent must have a positive delta mass Delta Mass Delta mass is the mass difference of an amino acid residue after it has been modified by the reagent being specified Applies to This attribute represents the amino acid s that this particular modifier can apply to In the case of reagents applying to sidechains these represent the modified residues themselves For terminus modifications any reagents specified will limit the modification to termini with an appropriate residue at the terminus An example of this is pyrrolidone carboxylic acid N TERM which can only occur on N termini adjacent to a glutamine Fragments The space separated masses and probabilities of any fragment ions resulting from this modifier reagent 3 To save the new or edited modifier reagent click the S
56. monitored in the bottom right corner of the ProteinLynx browser Specify the type of data table you wish to generate from the analysis EMRT Exact Mass Retention Time processed data e Proteins results of searches Depending on the other options selected for your experiment this section can display options for specifying which normalization method to employ in the analysis Automatic Internal Standards or no normalization If you wish to use Internal Standards select the boxes beside the standards you want to use If you do not wish to use normalization at all clear the Use Normalisation box The Go button is enabled when Apply is clicked in this section Starting an Expression analysis Once the Expression analysis is configured in the Design Manager the GO button becomes available Click GO to start the analysis Result Once the analysis has completed the tables specified in the Quantitation Analysis section are displayed The quantitation can take some time progress can be monitored in the bottom right corner of the ProteinLynx browser 10 9 Viewing Expression Results Expression results are automatically displayed when quantitation is completed To display existing Expression results 1 Expand the Expression experiment node 2 Expand the Expression Analysis Result node 3 Click EMRT Table or Protein Table and then right click 4 Click Open Expression Table EMRT table The table
57. name of an existing workflow template in the current project or the path to an XML workflow template file Processing Parameters Template The name of an existing processing parameters template in the current project or the path to an XML processing parameters template file Specifying samples vials and plates with Container Manager Optional recognized sample list columns Column name Description Parent Sample The presence of two or more Parent Sample columns indicates that the sample referred to in the Sample Name column is a processed sample This column can contain the name of a sample in the current project or a new sample Any sample attribute that appears and is modifiable in Sample Manager see Annotating and tracking samples with Sample Manager on page 4 1 can be specified through the inclusion of a column in the sample list Example If an imported sample list includes a column named Time Point the Time Point attribute of any sample specified in that sample list is set to the value in the sample list column Any column header that does not match a sample attribute or one of the column headers in the tables above is interpreted as a custom value Custom values are associated with the sample and can be viewed and modified using Sample Manager Example custom values in Sample Manager Custom Values Day Tuesday Month March Tap NONE DatabankLinks Custom Values
58. not available when viewing ion probability data To display a close up of a selected region of the graph in a separate window 1 Double click the Spectrum Viewer A red box on the graph indicates the selected region A separate window displays a close up of the selected region Zoom View To alter the size and position of the selected region To alter the size of the selected region click on an edge of the red box and drag to adjust the size of the box Results browser 6 25 To select a different region click inside the red box and drag to move to a different region Tip The close up window updates automatically as the size or position of the selected region is adjusted 2 To close the separate window and remove the red box from the main graph click the X in the top right corner of the separate window Copying data To copy the spectrum data or ion probabilities data click the 5 button to the right of the spectrum view Copying spectrum data If the spectrum viewer is showing a graph of the spectrum data the data on the clipboard is arranged to show a paired X value and Y value on each line The format is lt X value gt lt Y value gt Copying ion probabilities data If the Spectrum Viewer is showing ion probabilities a list of mass errors and influences is copied to the clipboard for each ion series that is being displayed The top line of the copied data shows the name of each ion series s
59. of protein sequences considered in the search Therefore Probability of A in mixture GIVEN dataset SUM over Probabilities of Combinations Containing A SUM over Probabilities of all Combinations Only the highest scoring peptide match is reported for each submitted precursor ion and its associated fragmentation data Where more than one peptide matches the data equally well for example if two peptide matches differ only by one or more isobaric residues all are reported B 5 How do know if a hit is real B 6 To determine if a hit is real always look to the top scoring protein Look at the spread of scores if the scores are grouped together they will have the same share of the available probability In practice given the variable quality of data the difference between the top score and the next highest score is usually a good indicator of the correctness of the highest scoring protein A difference of five factor of 150 is normally sufficient to indicate that the top scoring protein is correct Alternatively a proportion of the available probability can be assumed to be significant for example 95 For a database with 100 000 entries the maximum score would be 11 51 and the corresponding 95 significance threshold would be 11 46 1n 100 000 In 0 95 A difficulty can arise with the above criteria when a collection of largely homologous proteins get the top scores The available probability is then
60. option to TRUE This will make available further options relating to automatic downloads automatic updates and keeping of archives Select True or False as required Requirement When a new version of a databank is downloaded any workflow templates that relate to the databank must be updated with the new version number Periodically Download To periodically download the databank from a remote location set this option to TRUE Rule This attribute is only available if the Management Options attribute is set to TRUE If this attribute is set to True you must specify a remote location URL from which the databank will be periodically downloaded Download URL Address field There are several other options relating to periodic downloading that can be set or be left at their default values These are Download Compression Type e Download Renew Period Keep Archives Processing Start Time e Processing End Time 13 7 Databank attributes Continued Attribute Download URL Address Description Rule This option is only available if the Periodically Download attribute is set to True You must set this if the Periodically Download attribute has been set to True This field contains the URL address from which the databank should be periodically downloaded 1 Click the URL button and then type the URL address in the URL field 2 Click Open on the URL Chooser The system locates the re
61. or Mascot search engines Databank Search Databank Search Parameters Search Engine Type Mass Spectrum Peptide Tolerance 100 ppm Fragment Tolerance Estimated Calibration Error 0 1 Da 0 025 Da Molecular Weight Range Oto 200000 Da pl Range Minimum Peptides to Match Oto 14 1 Maximum Hits to Return 20 Primary Digest Reagent Trypsin Secondary Digest Reagent Missed Cleavages None Fixed Modifications Variable Modifications Exclude Masses Validate Results Yes Monoisotopic or Average Monoisotopic Ma Peptide Charge MH Instrument Type Search Engine Type Default Select the type of search engine you wish to use for this databank search Search Engine Type PLGS ki PLGS attributes 14 4 Query Tools Databank Search Databank Search Parameters Search Engine Type Data File MASCOT None Peptide Tolerance S Tolerance Estimated Calibration Error 0 025 Da Protein Mass 0 to 200000 Da pl Range Minimum Peptides to Match Oto14 1 Maximum Hits to Return 20 Enzyme Trypsin Secondary Digest Reagent Missed Cleavages None Fixed Modifications Variable Modifications Exclude Masses validate Re Yes Monoisotopic or Average Monoisotopic Mass Values Peptide Charge aa Instrument Type Search Engine Type Defaul
62. page 15 4 and Searching parameters on page 15 5 and then click File gt Save to save the parameters Rule Parameters cannot be saved if an acquisition is in progress In MassLynx click the start button to start the acquisition See also Refer to the MassLynx Help for assistance on starting an acquisition Click the Status icon to display search results during an acquisition 15 9 Real Time Status page with search results ProteinLynx Real Time Database Searching Of x File RealTime Settings Help Real Time Status 0 MassLynx Acquiring RT 5 03 Raw File C biolynx bin Real Time TestO01 raw MSMS Processing Submitted Queries 19 Proteins Excluded 1 Protein Excluded HXKA_YEAST 1 No 89 189 Hexokinase EC 2 7 1 1 Hexokinase PI Databank Searching HXKB_YEAST 8 Yes 628 646 Hexokinase B EC 2 7 1 1 Hexokinase PII HxK_DEBOC 1 No 64 078 Hexokinase EC 2 7 1 1 Setting up your DDA file Real time databank searching is designed to work interactively with DDA For this combination to work effectively the instrument needs to use de isotope peak detection and for this to work properly modifications to your DDA experiment need to be made The following graphic shows suggested settings for the Peak Detection and Exclude tabs of the DDA Survey experiment settings Exception On some instruments the settings shown below will appear in slightly different locations
63. page 5 25 Merging MSMS spectra and results If a sample has been separated into several fractions prior to being mass analyzed such as in a 2D LC or MudPIT experiment it can be preferable to merge the results that are generated from these fractions See also For further details on samples see Annotating and tracking samples with Sample Manager on page 4 1 To merge MSMS spectra and results 1 Select the required wells or spots and then right click 2 Click Merge Results 3 Select the sample for which the results need to be merged Only those samples that are associated with two or more of the selected positions are listed the default sample is never included These positions must also contain workflow results generated from Q Tof MSMS data Rule For positions with more than one set of completed workflow results the most recent will be included in the merge Results Ifthe sample selected is associated with a vial the merged workflow results and data will appear beneath the appropriate vial icon If the 5 24 Specifying samples vials and plates with Container Manager selected sample has no associated vial a new one will be automatically added to the current project to act as a place holder for the merged spectra and results The title for the merged results and data is automatically generated and contains the time and date of the merge action The results themselves will be displayed in a
64. peaks with SuperTrack 5 26 Interfacing with MassLynx 5 29 Troubleshooting failed client server workflows 5 33 5 1 What is Container Manager Container Manager can be used to Import lists of samples that you want to process using PLGS and associate raw data with the samples in those lists Assign raw data to samples that are attached to vials or plates the data can be processed searched and viewed using the PLGS results browser Chapter 6 Viewing results in the Results Browser Export sample lists to MassLynx see Exporting a sample list to MassLynx on page 5 29 the data is acquired in MassLynx see Acquiring data on page 5 31 and the results viewed in the PLGS results browser See also For an explanation of what the term sample means within PLGS and how samples are used see Chapter 4 Annotating and tracking samples with Sample Manager To open Container Manager click the Container Manager icon in the tool tray Workflow templates and Processing parameters The following sections refer to workflow templates and processing parameters Workflow templates used to perform an automated databank search of samples e Processing parameters determine how the raw spectrum data are processed and whether certain attributes for example smoothing are considered For more information on these concepts including information on how to create your own workflow templates and processing par
65. progress of the files being copied to the system A success message is displayed when the installation is complete Reboot the machine to ensure that environment variables are setup by the installer The following SYSTEM environment variables are created s LIBPATH lt installation path gt lib a PLGS_HOME lt installation path gt Configuring PLGS on UNIX When the installation is complete to configure PLGS for your specific system you need to Set the number of processors in the mkconfig file see Databank searching on page 1 25 Allocate RAM to the search engine see Search engine memory allocation on page 1 17 Create a TMPDIR environment variable see TMPDIR environment variable on page 1 18 Set a temporary directory for the search engine see Search engine temporary directory on page 1 18 Restore old databanks see Restoring old databanks on page 1 28 Search engine memory allocation When using large databanks with PLGS on a UNIX system you must alter the amount of RAM allocated to the search engine You do this by editing the ProteinLynx_SE startup script which is found in the bin directory of the installation Requirement Ensure that you have a minimum of 1 GB of RAM before changing the allocation To change the memory allocation 1 Edit the ProteinLynx_SE startup script from jre bin java Xmx256mb to jre bin java Xmx1024mb 2 Save and close the file TMPDIR environment variable
66. root root 4096 Jan 24 2003 lib 2 root root 4096 Jan 24 2003 libexec 1 root root 188561842 Sep 5 09 32 PLGS2 1_SP1FullLinux bin 2 root root 4096 Jan 24 2003 sbin 4 root root 4096 Jun 3 15 28 share 2 root root 4096 Jan 24 2003 src root localhost local PLGS2 1_SP1FullLinux bin InstallShield Wizard Initializing InstallShield Wizard Preparing Java tm Virtual Machine Running InstallShield Wizard Tip Use the ls l command to list all the files and directories and their current permissions in the current directory Run the binary file using the command PLGS2 2 5 INTEL LINUX bin or for SUSE Linux systems PLGS2 2 5 PPC_LINUX sh Result The ProteinLynx Installer dialog box opens Specify or browse for a directory in which to install PLGS Recommendation Install the PLGS in the directory usr local The default directory is usr local PLGS2 2 5 Specify the computer s IP address If needed use the ifconfig command to find the IP address 1 Open a terminal window Installing ProteinLynx Global SERVER 2 In the terminal window type ifconfig ifconfig command V root localhost Eile etho lo View Terminal Go Help root localhost root ifconfig Link encap Ethernet HWaddr 00 03 47 D8 A4 A3 Beast 10 62 255 255 Mask 255 255 0 0 UP BROADCAST RUNNING MULTICAST MTU 1500 Metric 1 RX packets 7098 errors 0 dropped 0 overruns 0 frame 0 TX packets 6 errors 0 d
67. samples to groups using the variables and values defined in Manually Define Experiment Variables Rule This section applies only if Manually assign sample groups is selected in the Select Grouping Method section To assign samples to groups 1 Click the Variable drop down box and then click the variable you wish to group by 2 Click the Value drop down box and then click the value appropriate for the samples you wish to assign 3 Inthe Available Samples box click the sample you wish to assign More than one sample can be selected using the Ctrl and Shift keys 4 Click the gt gt button to add samples to the group Result The selected samples are added to the Samples in Group box They are made unavailable in the Available Samples box and cannot be added to another group Select Data The Select Data section shows the processed data associated with the samples identified in the previous sections The first table contains a row for each sample the second table contains a row for each replicate associated with the selected sample 10 7 To show the attributes for a group 1 Inthe group table click the header of the third column 2 Inthe drop down list click the attribute you wish to display To select data for inclusion in the experiment 1 Click a group to see the associated replicates 2 To include the replicate in the experiment select the box in the Include column To exclude the replicate clear
68. see page 10 10 but does not contain columns for Cluster Include Average Mass Average RT Peptide or Probability Filtering the results To make the results easier to interpret or to reduce the size of the list in preparation for printing you can generate new results tables filtered by various criteria 10 13 To filter the results 1 Click the Filter button Nel 2 Type a title for the results table that will be generated for the filtered results 3 Set the filtering options as required see Replicate filter on page 10 14 Confidence Limit P value and Ratio filters on page 10 15 and Additional Filter settings on page 10 15 4 To see the data that will be included in the filtered results in the Log Plot Viewer see page 10 18 click Preview To generate the filtered results table click OK Result A new table is generated containing the filtered results A node will be added to the navigation tree below the results table that has been filtered Example filtered results tree FAL ARENE gpl 98 P certainty L FEB Accepted only EAN Protein Table Rule EMRT and Protein tables including tables containing filtered results cannot be deleted Replicate filter The Replicate filter enables you to limit the results to a specified number of replicates per sample To set a replicate filter 1 Select Use Replicate Filter Settings 2 For each sample set the maximum number of replicates that you
69. set thresholds manually to reduce the number of ions reported or to attempt to improve sensitivity Threshold The total number of ions not the height that the first peak in an isotope cluster usually referred to as the C12 peak must possess for the threshold criterion to be exceeded in a single scan Tip To estimate this centroid a typical scan containing analyte in MassLynx and look for this peak in a small but well defined isotope cluster Increasing the threshold can dramatically speed up processing by reducing the apparent complexity of the data Select time range Whether or not to limit by scans or retention time the range of data that should be processed Select start time The retention time at which processing should start Select stop time The retention time at which processing should stop Range Units The units in which the Time Range is specified 8 16 Creating custom processing parameters Viewing and processing gel data with Gel Manager Gel Manager lets you view and process gel data with clear sample tracking from gel to sequence identification Contents Topic Page Getting started with Gel Manager 9 2 Adding and importing data 9 3 Processing data 9 8 Viewing gel data 9 9 Getting started with Gel Manager You can perform various operations with Gel Manager Gels and cut lists lists of gel spots can be imported from a project or sample list into a p
70. subtype corresponds to the sequences in the flat file Formats are STANDARD NCBI_EXPASY_STANDARD NCBI_PRF_PIR NCBI_PDB NCBI_PATENT NCBI_GENINFO NCBI_GENERAL NCBI_LOCAL PDB PIR SRS ARABIDOPSIS_GENOME NRDB UNIGENE STANDARD_SPACED LONG_DESCRIPTION ACCESSION_ONLY UNKNOWN If the format is not FASTA this field is ignored Requirement In order for search results to contain accession numbers and therefore be suitable for protein quantification in Expression Analysis the FASTA format must be set correctly See also For definitions of the FASTA formats see FASTA flat file format on page E 9 13 5 Databank attributes Continued Attribute Location Description This field is compulsory Enter the file path of the flat file where the databank flat file is located When a databank has been created and saved this field cannot be changed If there is already a flat file of sequences for the databank use the File dialog box to choose this file If there is no flat file yet in existence for this databank and if the databank will be automatically downloaded choose the location to which the databank should be downloaded Requirement If the databank resides outside of the PLGS installation directory the Windows users who will run PLGS must have read write and modify access to the databank directory This requirement is especially relevant if the user adding the databank is an ad
71. text box 7 To save the template click on the toolbar Editing workflow templates Workflow templates can be edited using the cut copy paste and delete options available by right clicking in the workflow template panel or by using standard Windows keyboard shortcuts 7 9 Rule Editing the last search method on a branch will edit only that method However editing any other search method will affect all the results returned below it Opening workflow templates Workflow templates are saved as XML xml files and can be opened either from folders or from a URL To open a URL 1 Click File gt Open URL or click on the toolbar URL Chooser dialog box Paths Files URL Open Remove Cose 2 Specify the address in the URL field 3 Click Open A list of previously opened templates will be listed in the Paths and Files fields each time the dialog box is reopened 7 140 Defining templates for searching with Workflow Designer Filters When several searches are chained together the results of one search are filtered before being submitted as a query by the next search For Databank Searching AutoMod Analysis and De Novo Sequencing you can define this filtering process by specifying an XSL eXtensible Stylesheet Language style sheet XSL is a World Wide Web Consortium W3C standard defining style sheets for and in eXtensible Markup Language XML files lt Th
72. the navigator tree The new Processing Parameters Template is Changed in the Raw Data Spectrum Node Added to the Processing Parameters Templates node at the bottom of the navigator tree 5 21 Exporting and importing mass spectra PLGS exports and imports mass spectra in XML file format Exporting mass spectra Any processed spectrum can be exported To export a processed spectrum 1 Click a processed Mass Spectrum node and then right click and click Export Spectrum 2 Type an appropriate file name 3 Click Save Importing mass spectra Mass spectra saved as an XML file can be imported into PLGS To import a mass spectrum 1 Click Mass spectrum data not yet obtained in the navigator tree Figure titled Navigator tree Mass spectrum data not yet obtained on page 5 13 and then right click 2 Click Import Mass Spectrum 3 Browse to an appropriate XML file and then click Open Result The icon on the Mass Spectrum node changes indicating that processed data is now attached to it see the table Figure titled Navigator tree processing icons on page 5 18 5 22 Specifying samples vials and plates with Container Manager Working with plates There are several options available in pop up menus for target plates and microtitre plates Many of these are the same as the options available from the Container Manager navigator tree The available options are the same for target pl
73. the protein s contained in the original sample AutoMod Analysis tool Increases protein coverage and reduces unmatched MSMS spectra by taking the protein sequences identified through databank searching and rigorously analyzing these against the submitted spectra De Novo Sequencing tool Enables you to determine the primary sequence of a peptide directly from its MSMS data BLAST Basic Local Alignment Search Tool Searching tool Performs a homology search on the selected databank using the input protein peptide sequences Use these tools to create and edit individual queries submit those queries to the search engine and view the query results Contents Topic Page Databank Search tool 14 3 AutoMod Analysis tool 14 14 De Novo Sequencing tool 14 19 BLAST Searching tool 14 23 14 1 Query toolbar All the query tools share the same toolbar buttons Query toolbar buttons Button Description gt Submits the current query to the search engine View and edit preferences 14 2 Query Tools Databank Search tool The Databank Search tool enables you to search spectrum data against a protein or EST databank that has undergone a theoretical digest This search enables you to identify the protein s contained in the original sample You can perform the following types of databank search e PMF Peptide Mass Fingerprint PMF Fragmentation Ion Search Fragment Ion Search Using
74. the spectrum data does not include retention times Y axis intensity Each peak is labeled with peak mass You cannot directly select results in the Spectrum Viewer However the viewer responds to selections in the other browser components and colors the peaks in the spectrum to indicate the type of peptide Ifa protein or EST is selected the peaks that have been matched to peptides belonging to the selected protein or EST are colored If a mass is selected the corresponding peak is colored Ifa peptide is selected the peak that is matched to the selected peptide is colored The colors in the graph are Gray The peak is not matched to a peptide from the current protein or EST Blue A standard peptide that is with no modifications or missed cleavage sites Red A peptide that contains one or more missed cleavage sites Green A peptide that contains one or more post translational modifications Results browser 6 17 Yellow A peptide that contains post translational modifications and missed cleavage sites Viewing raw data i M f To view raw data click the button to the right of the spectrum view The processor needs to be running for the raw data to be retrieved as PLGS needs a live link to the raw data Result A two dimensional representation of mass X axis against intensity Y axis is displayed for the currently selected mass or peptide Raw data display w
75. this tool the search type performed is dictated by the type of mass spectrum data attached Databank Search details Type of Databank Search Type of Mass Spectrum Data PMF Maldi MS or Maldi Q Tof MS PMF Fragment Ion Search Maldi Q Tof MSMS Fragment Ion Search Electrospray Q Tof MSMS or Maldi PSD However using the Workflow Designer you can generate workflow templates that allow any type of Databank Search to be applied to any type of Mass Spectrum Data Example Use a PMF search for Electrospray Q Tof MSMS data Also using the Workflow Designer a Databank Search can be incorporated into a workflow as the first step in a more comprehensive analysis see Chapter 7 Defining templates for searching with Workflow Designer Databank searches can be submitted not only to the ProteinLynx search engine but also to a Mascot version 2 0 or later search engine The results can be displayed in the ProteinLynx browser or an Internet browser To open the Databank Search tool click the Databank Search icon in the tool tray 14 3 The Databank Search Parameters table opens in the Editor Panel of the browser with the Search Engine Type attribute highlighted The MASCOT option is available only if you have a valid connection to a Mascot search engine For details of how to connect to a Mascot search engine using the Preferences dialog box see Search Engine tab on page 2 5 Databank Search parameters for PLGS
76. types Mean Smoothing 8 10 Smoothing Window attribute 8 10 species 14 6 Species for Indexing databank attribute 13 7 specifier 12 10 Specify Plates dialog box 9 4 specifying estimated calibration error 14 21 maximum hits 14 21 maximum substitutions 14 16 processing parameters 5 15 substitutions and modifications per peptide 14 16 templates 5 15 workflow templates 5 15 spectrum icons 5 18 viewing 5 19 Spectrum Output tab 2 20 Spectrum Viewer 6 3 MS Data 6 16 MSMS Data 6 21 Options 6 24 SPTREMBL flat file format E 3 Start Sample List Run dialog box 5 31 A 11 A 21 Start Time 13 10 starting Expression experiment 10 9 MassLynx Acquisition 15 9 modules manually on Linux 1 13 on UNIX 1 19 on Windows 1 6 M9 sample list acquisition 16 8 PLGS on a client 1 5 PLGS on a single PC 1 6 PLGS on AIX 1 19 static bookmark 2 11 Subtract parameter 15 5 summary scoring B 2 SuperTrack 5 18 5 26 exporting results 5 28 Swiss Prot E 3 switch lists export 10 23 T table EST 6 12 tabs Search Engine 2 5 Spectrum Output 2 20 Tabular Data 11 14 target plate creating new 5 9 taxonomy 14 6 templates specifying 5 15 test procedure MALDI A 5 text filter print templates 11 16 threshold homology B 7 identity B 7 Index 17 Threshold attribute 8 13 8 16 Threshold Type attribute 8 7 tick OK column 6 12 Tof MS tab 16 5 TOF Resolution attribute 8 14 Tool Tray adding and removing tools 2 4 description 2 3 scroll buttons
77. want to be included for that sample in the filtered results You can either type the limit directly in the Number of Replicates column or use the up and down arrows to increase or decrease the limit Tip To specify the same number of replicates for each sample click the number in the Set the Number of Replicates in all drop down list 10 14 Using Expression Analysis to compare and analyze sample groups Confidence Limit P value and Ratio filters These filters enable you to return only those results that fall within set limits for the standard deviation of the log ratio probability of upregulation or ratio To set a confidence limit standard deviation of the log ratio filter 1 Select Use Confidence Limit Settings 2 Type a limit in the Ceiling box or drag the slider to set a limit To set a probability of upregulation P value filter 1 Select Use P gt 1 Settings 2 Type values in the boxes or drag the sliders to set the limits Clusters with P values between the Floor and Lower and clusters with P values between the Upper and Ceiling are included in the filtered results To set a ratio filter 1 Select Use Ratio Settings 2 Type values in the boxes or drag the sliders to set the limits Clusters with log ratios between the Floor and Lower and clusters with log ratios between the Upper and Ceiling are included in the filtered results Additional Filter settings There are a number of additional ways of filter
78. ways In the Container Manager navigator tree click the workflow results not the workflow template and then right click Click Print In the results table click a protein and then right click Click Print Workflow 11 6 Creating print templates and printing project data Workflow print wizard pop up menu in Container Manager navigator tree Container Manager te Hans Aerts m AMC Hans Aerts Ce j B vials ee Workflow Results 20 proteins Probability rota Plates Pe wil i e AGAL HUMAN 478 447 late one E AMG Hans aits niilc Q91293 Carbamoyl phosphate ia nille Q23495 Hypothetical 185 2 Ad ia el 10 42 2 50 C AExpres nille QIQUN7 Toll like receptor a rn D ulile P55123 Leukotoxin BE AmC Hans Aerts i Submitted Mass Submitted Charge ill 10 42 2 50 CAExp ille 12495 Chromatin assembly 3 669 x Processing Pa gt nille Q97WVHO DNA double strand b gt c1 gt c2 gt D1 gt D2 E Plate2 EA Target Plates Processing Parameters Template S23 Workflow Templates 77063 02 max Precursor mass 1413 6687 charge 1 Ler TM H GWLHWER H bMax REH wHHiLE wH m E et yMax A TE 0p wes 11 ot 1 41413668 E pe 228 159 1 tgo 1000 74 yMax yi 708 o 1000 DECCAL 462 1703 max Workflow print wizard pop up menu in a results table Container Manager AMC Hans Aerts Hg6 Vials iicroiira Pistes Workflow Results
79. when the Databank Admin Tool opens try restarting the search engine For help with starting modules see Chapter 1 Installing ProteinLynx Global SERVER 13 2 Organizing databanks with the Databank Admin tool Adding databanks To add a new databank 1 Click B on the toolbar or click File gt New Databank A Databank editor panel opens under the navigator tree Databank editor panel and navigator tree Databank Admin Tool Databank PROTEIN NCBI_EXPASY_STANDARD None Make Blastable TRUE Index For PepGrab FALSE Load into Memory FALSE FALSE FALSE None FALSE None Set a suitable name for this family of Databanks Name 2 Tochange the values of any attributes click the attribute in the panel and then edit the value under the panel See Databank attributes on page 13 4 for details of the attributes and values 13 3 3 Click to save the new databank The new databank is displayed in the navigator tree If the file is large processing of the databank file can take several seconds When the file has been processed the databank is available to the various Protein Probe tools and the databank name is displayed in the Databanks field of the Databank Search Tool To ensure that the most up to date state of the Databanks are being displayed in the Databank Admin Tool click Refresh Databanks Tree View on the toolbar Datab
80. within the experiment dialog box Refer to the MassLynx Help and the Operator s Guide for your instrument using the settings below as guidelines 15 10 Real Time Databank Searching Peak Detection tab Function 1 Survey Scan Adducts Collision Energy LockSpray Variable Flow Acquisition MS Survey MS MS Peak Detection Exclude Include m Peak Selection Apply no criteria other than intensity as specified on MS Survey tab for peak selection Charge State Peak selection Isotope Pattern selection V Deisotope Peak selection Peak Detection Window 4 Da Charge State Peak Selection Select Charge States of interest 1 2 3 4 5 B Use advanced options for Charge State peak selection r Deisotope Peak Selection IV Use advanced options for Deisotope peak selection Tolerance Window 35 Da Peak Extraction Window Z Da De isotope peak detection For a more in depth description of the workings of Deisotope Peak Detection see the MassLynx Help De isotope peak detection is enabled by selecting the Deisotope Peak selection box on the Peak Detection tab of the DDA experiment settings Figure titled Peak Detection tab on page 15 11 15 11 Tolerance window The tolerance window is a window of user defined m z that slides up the m z range looking for isotope clusters Only peaks that are above the intensity threshold are considered in this routine An ideal value for this is the dist
81. workflow results window and have the same format as a single set of workflow results The merged workflow results will not contain duplicate proteins but all the submitted masses will be included even if they are duplicated Customizing the plate view To modify the colors of the plate view 1 Click Options gt Preferences gt Colours tab For further details see Colours tab on page 2 12 5 25 Simplifying peaks with SuperTrack Rule SuperTrack is only available for MS data The SuperTrack tool enables you to validate your raw data before performing databank searches It looks for replicate EMRTs Exact Mass Retention Times and reports only those peaks that have the same m z and retention time for all three replicates Further the high energy peaks must associate with the same precursor in all three cases The simplified spectra can accelerate databank searching and improve protein identification This can be particularly beneficial if you intend to perform databank searching using Mascot as Mascot prefers fewer peaks See www matrixscience com for more details about Mascot Requirement Processed data must include retention time information to be compatible with SuperTrack Data processed with PLGS versions prior to 2 2 5 does not include retention time information To open SuperTrack 1 In the tool tray click Container Manager 2 Open a ProteinLynx Global SERVER project by clicking the Projects drop down b
82. you start the modules automatically by starting the ProteinLynx browser log files are generated by the software These log files can help you to solve operational problems and will be helpful to Waters if you request technical support To view log files 1 Navigate to the PLGS installation directory and then to the log subdirectory 2 Open the log file in a text editor such as Notepad Two log files are created Processor txt for the processor log SearchEngine txt for the search engine and microkernel log 1 6 Installing ProteinLynx Global SERVER Installing PLGS on Linux This section describes the steps required to install and run PLGS on Linux PLGS can be installed under Red Hat Linux 9 on Intel based architectures or SUSE Linux Enterprise Server 9 on IBM Power architectures On Linux the ProteinLynx browser enables you to add new databanks to the server or view online help see Linux ProteinLynx browser on page 1 18 Restriction Only the Databank Admin Tool and the online Help are available in the Linux PLGS browser If so configured processing and searching can be run on a Linux machine from a remote Windows PLGS browser Rule All UNIX commands are case sensitive Before installing PLGS Complete these tasks before installing PLGS in Linux Back up the PLGS directories see Backing up the PLGS folders on page 1 7 Ensure that you are logged on with root permissions see Changing file
83. 0 ppm lockmass Molecular Weight Range PLGS or Protein Mass MASCOT These attributes are optional as a default range is supplied Restriction This cannot be used for searches of EST databanks This attribute restricts the number of returned protein matches to a range of molecular weights PLGS or masses MASCOT Specify a narrow range to reduce search times Tip The range could be based on the location of the gel from which the sample that generated the data originated When looking for a specific protein of interest the size and range indicates the confidence in the estimation of the molecular weight or protein mass For a PLGS search type the minimum and maximum molecular weights in Daltons For a Mascot search specify the maximum protein mass in Daltons pl Range This attribute is optional by default all proteins are searched This attribute restricts the number of returned protein matches to within a specific iso electric point range The range could be based on the location of the gel from which the sample that generated the data originated or the range of a specific protein of interest Using a narrow range reduces search times Restriction This attribute cannot be used for searches of EST databanks or for Mascot searches To specify the range type the minimum and maximum iso electric points in the text fields 14 8 Query Tools Minimum Peptides to Match This attribute is optional as a default
84. 00 Use this with the Toggle grid function to display pages across the display as in the graphic 11 11 Opening and deleting print templates The same dialog box is used to open or delete existing templates whether they are default or user defined To open or delete an existing template E 1 In the tool tray click to open the Print Tool 2 Click Hd Alternative Click File gt Open 3 Click the template name 4 Toopen the template click Open To delete the template click oO 11 12 Creating print templates and printing project data Creating print templates Use the Print Tool to create project or workflow templates The templates you produce are displayed as user defined templates in the Project print wizard or Workflow print wizard See also e Project print wizard on page 11 3 e Workflow print wizard on page 11 6 To open the Print Tool click the Print Tool icon in the tool tray To create a new template 1 Click E3 Alternative Click File gt New 2 Type a name and then click Next Print Tool New Template amp New Template ProteinLynx Renderer Select the type of template to render with Please select the representation of the data Tabular Data Graphical Data Select this if you are creating a workflow template EAE 11 13 Select either Graphical Data or Tabular Data 4 Choose a setting for the Support workflows only check box e For a template to pr
85. 2 22 pl range 14 8 plain text files txt 9 4 plate colors defaults 2 13 Plate View 5 23 PLGS folders backing up in Linux 1 7 backing up in Windows 1 3 PLGS search engine 7 6 PLmicokernel 15 15 PlugIn Selector dialog box 2 25 PlugIns Export 2 23 adding 2 24 Import 2 23 replacing 2 24 preferences changing 2 5 previously acquired data processing A 2 primary digest reagent 14 9 14 16 14 21 Primary Internal Lock Mass attribute 8 7 print templates curated filter 11 16 deleting 11 12 numeric filter 11 16 opening 11 12 text filter 11 16 Print tool 11 2 11 25 description 11 2 Print Wizard 6 16 10 13 11 3 print workflow 6 16 printing 11 2 Expression results 10 13 opening and deleting templates 11 12 project template 11 2 results 6 16 templates 11 2 workflow template 11 2 probability of upregulation filter 10 15 Process Mass Spectrum 5 7 Process Method parameter 15 5 Process Raw Data 5 7 Process Raw Data command 5 17 process_kernel 15 15 processed spectrum 5 19 Processed Data Viewer 5 19 processed samples generating 4 5 processing Index 13 data from a sample list 5 7 Gel Manager 9 8 parameters 15 4 previously acquired data A 2 Processing End Time databank attribute 13 10 processing parameters 5 2 5 6 adding 5 21 changing 5 7 MALDI attaching A 8 Q Tof MSMS attaching A 18 setting A 14 setting A 6 specifying 5 15 processing parameters templates 5 21 8 5 attribute sets Chromatogram 8 5 8 15 Deisot
86. 20 proteins wile a ph e o E plate one E AMC Hans Aerts wile 91293 Carbamoyl phosphate 7 At wile 23495 Hypothetical 185 2 i wille QIQUN Toll like receptor wile P55123 Leukotoxin wile Q12495 Chromatin assembly m AMC Hans Aerts AA 10 42 2 50 C lExpressia AMC Hans Aerts it 10 42 2 50 CAExpres j wile Q97WHO DNA double strand b X Processing Parar z lil P93844 Phospholipase D 2 p c1 willie Q03410 Synaptonemal comple g c2 gt willie P01029 Complement C4 precu gt D1 gt D2 E Plate2 EFA Target Plates Processing Parameters Templates Workflow Templates 77063 02 max Precursor mass 1413 6687 charge 1 F te TM 4 GWLHWER bMax HREH w nHiL t wH mH PT ymax woi mj bit tt 1 1 1413 668 n e bieza pee A if Max 3 228 159 1 4 1000 748 yl 462 1703 max OROASIA Whichever method is used a template selection dialog box opens 2 Select to use either default templates or user defined templates and then click Next Recommendation New users should select default templates 3 Click a suitable template and then click Next 11 8 Creating print templates and printing project data Workflow print wizard Choose a Print Procedure Ja Print Workflow a Edit Limits Proteins content In this screen you can print immediately preview the report see Figure titled Previewing a Wor
87. 39 788 4561 788 4644 0 0083 E 0 0537 n P01011 Alpha 1 antichymotrypsin precursor ACT 880 4291 879 4213 879 4338 70 0125 3 0 2059 4 927 4847 926 4769 926 4861 0 0092 9 r 1 8469 l orre ra al al 933 5135 932 5057 932 5114 0 0057 6 1 6286 lile P02768 Serum albumin precursor X x a 718095 7 Counts a 2 lala lalale max 33 057 BAHE The results display enables you to select various different views of the data To view further details click individual results items The results browser is divided into four sections the navigator tree table of protein and EST data table of peptide data and spectrum viewer Each section can be resized by clicking and dragging the dividers Results browser 6 3 If the results are for MSMS spectrum data two spectrum viewers are included one shows the parent spectrum and the other shows fragmentation data Browser display of results for MSMS spectrum data mw 2515 Expression B 2 4 1 127 hits 194 proteins ES nille Serum albumin pre ane bald imal ee oe m P02787 E Serotransfertin precursor Siderophilin Beta 1 P nille Serotransferrin precursor Siderophilin i P01922 HBA_HUMAN Hemoglobin alpha chain P wille Hemoglobin alpha chain Q14473 Q14473 Beta globin gene from a thalassemia patient
88. 5 026 1 P02023 Hemoglobin beta chain B P00330 Alcohol dehydrogenase EC 111 0 SIGGEVFID A 0 07 2 62 0 65 0 0 P02768 Serum albumin precursor 0LDELRDE j BG P02647 Apolipoprotein Al precursor Apo Al 0 VOPYLDDF 5 21 i BG P29312 14 3 3 protein zeta delta Protein kin 0 DICNDVLS P04406 Glyceraldehyde 3 phosphate dehyar LISWYDNE AT 2 P01834 Ig kappa chain C region O HKVYACEV BG P00330 Alcohol dehydrogenase EC 111 O SISIVGSYV 1 88 0 63 0 21 1 00 0 15 1 91 0 79 0 0 P12110 Collagen alpha 2 VI chain precursor 0 GDPGNR _ 0 46 0 78 1 03 0 0 Po1009 Alpha 1 antitrypsin precursor Alpha DTEEEDF 0 23 1 49 0 16 0 00 AG P02768 Serum albumin precursor 100 0 KOQTALVEL 0 54 0 61 0 23 0 00 P01857 Ig gamma 1 chain C region 100 0 P21810 Bone cartilage proteoglycan prec zeto fefee fefta t foe ef aeae tae fee eeta faee e e COC COC P02787 Serotransferrin precursor Siderophi P02647 Apolipoprotein A precursor Apo Al OWOEEMEL P51884 Lumican precursor LUM Keratan s 0 SLEDLQLT P05092 _Peptidyl prolyl cis trans isomerase 0 VNPTVFFD 1848 886 Q04695 Keratin type cytoskeletal 17 Cytok O TMQALEIE 996 556 P02787 Serotransferrin precursor Siderophi ASYLDCIR 1300 754 j P12110 Collagen alpha 2 VI chain precursor 0 DIAST
89. 7 Using navigation and installation commands D 8 There are various commands that assist navigation and installation Commands to aid navigation and installation Command Description hostname Echoes the system name whoami Echoes the current user name pwd Echoes the current path location ls a Lists the contents of a directory cp Copies a file or files to another name or location cd Enables the user to change directory or example cd tmp changes from the current location to the tmp directory mkdir Creates a new directory in the current location chmod Changes the permissions of a file more Lists the contents of a file pg Lists the contents of a file UNIX Help for Installing PLGS on AIX Platforms r A r NNNN oO ff S gt Dp rf Use SMIT to create and manage user accounts and groups Setting the HOME Directory is very important A user s HOME Directory should never be the root directory D 9 The sequence of directories that commands search can be set for all users or for selected users For all users it should be included in the etc environment file and for selected users it should included in the user s HOME profile file Because the profile file is hidden use the s a command to list it Use the VI editor to edit these files It is advised to always make a copy of a file before editing For example cp environment en
90. 8 13 8 15 minimum peptides to match 14 9 missed cleavages 14 10 14 16 modifications to peptides specifying 14 21 modifier reagents adding 12 4 deleting 12 6 saving 12 5 viewing 12 3 Modifier Tool 12 2 12 6 Modifier type attribute 12 5 Modify Bookmark dialog box 2 12 Modify Processor dialog box 2 9 Modify Search Engine dialog box 2 7 modifying bookmarks 2 12 processors 2 9 sample 4 3 search engines 2 7 Modules starting manually on Linux 1 13 starting manually on UNIX 1 19 starting manually on Windows 1 6 molecular weight range 14 8 monoisotopic 14 12 masses 5 19 MS Data A 10 A 20 MS Method 5 30 A 5 A 14 MS Method Editor 15 3 MS Text format 2 22 MS data 7 2 7 5 16 1 function 16 2 method file 16 3 MSMS tolerance 14 7 multiple associated masses 6 35 fixed modifications 14 11 species 14 6 variable modifications 14 11 mzData format 2 23 N Name attribute 12 4 Index 11 Name databank attribute 13 4 NanoLockSpray 16 2 navigator tree 6 2 6 9 results browser 6 7 NCBI E 6 New Container Tool dialog box 5 9 new databank adding A 25 New Expression experiment 10 3 new project creating A 2 noise reduction Q Tof MSMS A 16 Noise Reduction attribute set 8 5 non specific digest reagent 14 10 normalization automatic 10 9 internal standards 10 9 NP Multiplier attribute 8 14 Number of Precursors attribute 8 15 numeric filter print templates 11 16 O OK column cross 6 12 question mark 6 12 tick 6 12
91. 814 USA COMMENT Method conceptual translation FEATURES Location Qualifiers source 1 101 organism Plasmodium falciparum 3D7 strain 3D7 db_xref taxon 36329 chromosome 2 product metal binding protein DHHC Protein 1 101 domain CDS 1 101 gene PFB0725c coded_by complement join AE001414 1 1256 1365 Similarity putative ORIGIN cnllkikrsh hcsvcdkcim AE001414 1 1500 1634 AE001414 1 1821 1881 note identified by sequence miiwchikcl ctnpgflnet fhfvsdntte ydnnvgqmckk 61 kmdhhcfwin scvglyngky fillnfvrtk gkyntniikh 1 E 7 BLAST flat file format This format is the same as the NCBI_EXPASY_STANDARD format subtype of FASTA format Example gt gi 3845261 gb AAC71934 1 metal binding protein DHHC domain Plasmodium falciparum 3D7 MIIWCHIKCLCTNPGFLNETFHFVSDNTTEY DNNVOMCKKCNLLKIKRSHHCSVCDKCIM KMDHHCEFWIN SCVGLYNOKYFILLNFVRTKGKYNTNIIKHL E 8 Databanks Formats FASTA flat file format FASTA format consists of a description line beginning with a gt symbol followed by multiple lines containing the sequence of amino acid or nucleotide characters Example gt gi 3845261 gb AAC71934 1 metal binding protein DHHC domain Plasmodium falciparum 3D7 MIIWCHIKCLCTNPGFLNETFHFEVSDNTTEY DNNVOMCKKCNLLKIKRSHHCSVCDKCIM KMDHHCEWIN SCVGLYN
92. 9 PASTAL EELPEF PIR paok he es E 10 FASTA MOBIL PDD arroa S E 10 PAS to BE PATENT order e A aes E 11 FASTA NCBI a F oana eso ean E 11 FASTA NCBI GENERAD oaunsorderras irii r TE E 11 eae PLEO CAL Do E 11 fee k ig Ck Manne em EE E enn E EEE my ely E On eerste terest amon Me Mer ee ee este E 12 ries NE ld See E EE A A A eee E ee eee meee eet on Ree ne eas ener E E 12 FTA E vic essere neta ee es E 13 PASTA ARABIDOPSIS GENOME ys cisssveissvcssesesesnvesvwsisssresdaeeenvertarsimdeier E 13 FASTA AC csser eee e ne rene esas AOR ane E 14 FASTA UNN EN ocean ene see egeee emcee A E 14 FASTA STANDARDU SPACED iciciiittesdseateiainininainannnuinsnnniewey E 14 FASTA LONG aicisiersrcitwncwencencaunuuaawunaien E 15 FASTA ACCESSION ONLY ves eccscciniicicccsian nares TEET E 15 TCO T E A E E E E E A E T Index 1 xviii Table of Contents Installing ProteinLynx Global SERVER ProteinLynx Global SERVER PLGS is a multi platform Java C and C application which features a new and comprehensive range of integrated tools for proteomics project management protein quantification and protein identification and characterization through exploiting the specificity of exact mass data ProteinLynx Global SERVER can be run in a client server environment or on a single PC When run on Linux or UNIX ProteinLynx browser contains the Database Admin Tool and Help This chapter describes the procedure for installing PLGS on the following platforms Each pa
93. A text file containing the switch list information for the selected clusters 1s created in the location specified 10 23 Importing Significant Clusters You can import a list of significant clusters into your EMRT results table to simplify and accelerate the process of selecting clusters for other operations such as exporting switch lists or searching EMRTs To import significant clusters 1 Inthe EMRT results table see EMRT table on page 10 10 click Import Significant Clusters al 2 Browse to the location of the clusters file you wish to import 3 Click the file and then click Open Result The Include column is selected for the clusters listed in the imported file Significant clusters list file format Significant cluster list files are plain text files containing one cluster number on each line Example 18 41 55 84 10 24 Using Expression Analysis to compare and analyze sample groups Assess Data Quality viewer If you are unsure whether your data is good enough for quantitation or if you find that your quantitation results are not what you expect you can view statistics for each injection in the Assess Data Quality viewer To open the Assess Data Quality viewer 1 Click the arrow at the right side of the Assess Data Quality section so that the panel is displayed Requirement You must have an Expression experiment open to do this See Experiment Analysis Design Manager on
94. Bookmark Ei Link from BLAST Results 7 PROT http fus expa false true bwiss PROT TrEMBL nttp 1us expasy orgiegi binige Adding a bookmark You can add static or dynamic bookmarks to the list To add a bookmark 1 2 3 Click Add to open the Add Bookmark dialog box In the dialog box type the name of the bookmark and the URL Select the Static Bookmark check box if the bookmark is static always the same or clear the Static Bookmark check box if the bookmark is dynamic A dynamic bookmark is not a valid URL until it is combined with a unique identifier For example to form a valid URL the SWISS PROT TrEMBL link that is supplied with ProteinLynx browser requires the addition of an accession number This URL then provides a link to the SWISS PROT TrEMBL databank entry for the specified accession number 4 Select or clear the Link from BLAST Results check box If selected hyperlinks to the external database can be formed from accession numbers returned from BLAST Basic Local Alignment Search Tool searches 5 Click OK to save the changes Modifying a bookmark You can modify the name URL static bookmark status and BLAST results link status of a bookmark To modify a bookmark 1 Double click the bookmark in the list Alternative Click the bookmark and then click Modify 2 The Modify Bookmarks dialog box opens which has the same fields as the Add Bookmark dialog box 3
95. CAAAGGT GGGCAGCTCTGAGACAATGGTGGTCAAGTGACCACTGAGGCCCAGAGCCGTTGGAACAGT CTCTTAGAACAGGGTGGAGGACTTAAAACTTGGATGAACAGGGGCTGGCAGAGCACTTGG AATGGGTAAGGACAAGACCGGGAGATCAATTTGGCTGGAGCAGGGGAGCTTGTGTTATAT ATGCAGAAAAAGGTTGAAACGGGGAAGTTTTAATACTGTTTAGGTAAATAAGGATTAAAC ACAAAAGGAAGGAAAAACGTGAGA FASTA STANDARD_SPACED Description line gt NAME ACCESSION NUMBER DESCRIPTION Example gt IF3 AQUAE 067653 Translation initiation factor IF 3 MSKLKEYRVNROQIRAKECRLIDENGQOIGIVPIERALKIABEEKGLDLVETAPOQAKPPVCK IMDYGKFKYELKKKEREARKKOREHOTEVKDIRMKVRIDEHDLOVKLKHMREFLEEGDKV KVWLRFRGRENI YPELGKKLAERI INE LSDIAEVEVQPKKEGNFEMI FVLAPKRKK E 14 Databanks Formats Description line gt NAME DESCR PT ON FASTA LONG_DESCRIPTION This format is used when the description is very long In the ProteinLynx display the description is truncated to fit into the viewing area Example gt gp AL034396 1 PID 5441319 Human DNA sequence from clone 1158B12 on chromosome Xp11 21 11 4 Contains the ZX for X linked duplicated Zinc finger A and MYCL1 avian myelocytomatosis viral oncogene homolog 1 1 carcinoma derived and KRT8 Keratin type STS proteins ME skeletal GSSs and a CpG island Sw P98168 Sw P98169 PKLLPARGTLOGGGGGGI EASTASRGPGPSLFAPRPH
96. DATA REQUIRED lt Ref_text Description This describes PCDATA of an element referred to by a reference Documents Query Attributes gt lt ELEMENT REF_TEXT PCDATA gt lt Url Description Describes a url Documents Query Project C 24 Implementing a plugin for ProteinLynx Global SERVER Attributes protocol the protocol host the hostname or ip address port the port number path the path gt lt ELEMENT URL EMPTY gt lt ATTLIST URL PROTOCOL http https file file HOST CDATA IMPLIED PORT CDATA IMPLIED PATH CDATA REQUIRED C 25 Plugin process exit codes The plugin process exit codes are Plugin process exit codes Code Description 0 Successful completion 1 File not found 2 Invalid query 3 Error 4 Busy C 26 Implementing a plugin for ProteinLynx Global SERVER UML Class Diagram for the PLGS plugin Architecture The following diagram illustrates the PLGS plugin architecture UML Class diagram for the PLGS plugin architecture UML Class Diagram of the PlugIn architecture run void PluginHandler mHandler PlugInHandler handleStart plugInInputStream OutputStream pluginOutputStream InputStream PlugIn h PlusinHandler Plugin pluginErrorStream InputStream boolean jee a re ae thandleOutput bytes byte n int boolean d handleError bytes byte n int boolean handleException e Excep
97. E ne tee ne ean ee reer E E E em wren eee terre A 11 Acquiring Q Tof MSMS Gata sicccicssccssscctessctessscscsssecesectesssustseceevsesssensesvescstesdss A 14 Setting the microtitre plate cccccecccsccssecceccsccsecceccceeeeeeeeeeeeeeeeeeeeseeeeeseeeess A 14 Setiine processing PAT AMICEETS criera aaa adeh aahi aiia A 14 Croa or aae R ON eo a a A 17 Attaching the data processing paraMetelLs cccccscccccccsssssssseceeeeeeeeseenaaees A 18 Attaching the workilow Tle vise ccieseccanticiawecssweseietanlaneeisaeceiawccesnioesearsiawvecnimecues A 19 Exporting the samyle list to Mass 06 cssis1sascesctecrsvieeacsssearsdessvearzonndeeeneass A 19 PON A OA iene E eho ee eet el eke A 21 Adding a new databank 0 0 0 ccsccscessscesccesccecccnccccccccccecccccccecccccceeccccccecceeees A 25 PE Scoring SCHEINGS sicsccistccenqrsinicacressdoinneraaineauntmamaiee B 1 MOCOLIM G SUMMALY sioe eaidece lek aah toacee la cen ata se skate a a a a eke B 2 MALDI scoring PMF PMF fragment ion searches ccceceeceeee B 4 MSMS scoring fragment ion searches essssseessesssesssssssosssosssossoossosssoosooesso B 5 How dol know ifa hitisreal ssissssisesossssisassisdssevessedsdisosrssodeaasiauisosoiesisas B 6 Automatic data Curation co occccssxs css cecsccacntcatdatatlecesacscasicecedevansatercuantsacacsteancss B 7 BR shoes A M E E A T Goes A E AE E E seen S A A T E ENT B 7 PMP Fragment lO eauso E aS B 7 HP Sei LOW a e B 8 Electrospray MS cccccccsssssscssstsaesinas
98. Example projects list CSF_225Processing x CSF_225Processing My project aters Micromass Project 1 PLGS2tTraining 3 Click a project to display it in the browser Project names in black text are available but not currently open Project names in blue text are currently open Project names in gray text are unavailable they cannot be opened Projects might be unavailable because they are currently being saved or deleted Updating projects When MassLynx is used to acquire data based on information exported from ProteinLynx Global SERVER PLGS projects can be updated to reflect the most recent information available Updating projects is not usually necessary at other times To update a project In the ProteinLynx browser click File gt Update 3 5 Closing and deleting projects To close a project 1 3 In the tool tray click the icon for one of the tools that requires a project Sample Manager Gel Manager Container Manager or Expression Analysis If the project is not currently displayed switch to the project you wish to close see To open a project on page 3 5 for details Click File gt Close Result The selected project is closed releasing any resources it is using and closing any associated windows Rule If changes have been made since the project was last saved you can save the project before it is closed To delete a project 1 3 4 5 In the tool tr
99. Expression Results oc ccs cccisscsccssssssssssceessaccsesssesssceesessesseoesevesessaseess 10 10 EMET Tahe co E E 10 10 Proen Abispa Ee Re 10 13 Piltar me he Pi Ea pars aR ea es yecsce ees odie ee 10 13 peer sey ana 6 Les age eee ee eee S 10 14 Confidence Limit P value and Ratio filters oo ceeececcseeeseseeeeees 10 15 Addiiional Filter tc1 ao Wy 71 gt gee eee ee ae ey ae ene ee en en eee cee 10 15 Toportine work TS 5c codes nececusdecandeces suc A E 10 16 Searching EMRTs from the EMRT table cee eeeeeeeeeeereteetteneteeeeees 10 17 Log Plot Viewer eercsiseesedesesvaceseeatesdesseecacesdad esate exeuwssecdccevessouseeseeuececescewteectees e 10 18 Expression Data Viewer cisiiissceccscesscsccesdsesssssesecsesscaecasdcceteasesassesessscessesesesss 10 20 Group eol ara O meee eios 10 21 CA E A A E E E es ees 10 21 Replicate Spectrum level sc aicsccsn sisi nee ranei 10 21 Table of Contents xi Exporting Switch Lists icsse issen tisanas noes eaaa aaaea a aaaea assaia 10 23 Importing Significant Clusters s ssssssssecsescssesceecessscssoecoscssccssecsscsossesscsosseo 10 24 Sienieantelusters hat ile TOMAT sroin n eeaeeeae 10 24 Assess Data Quality viewer cccccccccccccsccecccecccecceeccecccecceeccececeecsecescecseeees 10 25 11 Creating print templates and printing project data ccccee 11 1 Printime data oios act acenceseacestacci ees sactccasiesssessveaheseasscaustiansctansstaseeess 11 2 Usine print WIZALGS 5ssessesis
100. For this type of container Vial Do this 1 2 Click the vial you wish to set the sample for Right click and then click Set Sample Microtitre plate Target plate Click the microtitre plate you wish to set samples for Click a spot on the microtitre plate display Right click and then click Set Sample Click the target plate you wish to set samples for Click a spot on the target plate display Right click and then click Set Sample 2 Inthe Select A Sample dialog box click Default and then click OK Tip Sample Manager see Annotating and tracking samples with Sample Manager on page 4 1 enables you to organize and annotate your samples If you have already created samples in Sample Manager you will be able to choose them at this stage and then track and use them throughout your PLGS project Result A new node is added to the navigation tree below the container selected If a sample has been set for a microtitre or target plate spot the spot changes color 5 12 Specifying samples vials and plates with Container Manager Attaching raw data If a vial microtitre plate or target plate is being used the raw data must be attached manually If a sample list was imported the raw or processed data is already attached to those samples To select raw data 1 Inthe Container Manager navigator tree click the Raw Data Spectrum Node and then right click
101. LDIWF xml that you created earlier see Creating a workflow on page A 7 and then click Open Exporting the sample list to MassLynx For further details see Exporting a sample list to MassLynx on page 5 29 To export the sample list 1 In Container Manager right click on the target plate node and then click Export Sample List to MassLynx A 9 Export to MassLynx dialog box P7 Export to MassLynx Project MALDITestProcedure PRO Method File malditest exp Inlet File Default Tune File None File Name MALDI Test Listt Data Name sample i i Samples included in a previous list will not be included in this list nless data acquisition was cancelled before spectra were obtained e default injection volumes will be used for the exported samples nless the olb file is imported into MassLynx and the injection volume or each sample edited manually 2 Specify the MassLynx project from which the data is to be acquired 3 If more than one MS Method is stored in the MassLynx project use the drop down list to specify the correct file Tips The File name can be the same as the target plate name The MS Data name can be changed to any text such as digest_0 adh_0 4 Click Export 5 In MassLynx click File gt Import Worksheet 6 The file created by PLGS is stored in the MassLynx project Browse to the file and then click Open Result The MassLynx sample list
102. M Amidation C TERM Biotin K Carbamidomethyi C Carbamyi K Carbamyi N TERM Carboxymethyi C Click a reagent in the list to specify a variable modifier that should be applied to peptides produced by the digests To select multiple modifier reagents use Shift click or Ctrl click Both modified and unmodified versions of each peptide will be used in the search Validate Results All MSMS results can be validated A validated peptide will contain a series of three or more consecutive y ions If validation is selected the top scoring peptide for each MSMS spectrum is returned This could increase the requirement for manual validation of the results returned 14 22 Query Tools BLAST Searching tool The BLAST Searching tool performs a homology search on the selected databank using the input protein peptide sequences BLAST predicts which proteins the input sequence could be a part of BLAST searches can be performed as one off searches using the BLAST search tools BLAST searches can be performed using the workflow system enabling the BLAST search to be combined with other searches See sections on Workflow Designer page 7 1 and Container Manager page 5 2 for details of how to perform BLAST searches and other searches as part of an integrated workflow Tip Careful use of the algorithm through automated workflows can increase coverage and confidence of the top databank search hits while simultaneously filtering ou
103. OJECT gt lt REF_ATTRIBUTE NAME PROJECT_ID VALUE Project3 gt lt REFERENCE gt lt TAG gt lt PROJECT gt lt PROJECT gt lt TAG gt lt UPDATE gt lt QUERY gt Updating a Project document for a given Project_ID Update queries like select queries are done at the element level The insertion or deletion of an element within a document can be thought of as an update to the parent element Therefore an update comprises the location of the element to be changed or the parent element of elements to be deleted or inserted and the specification of its replacement if the element has a required attribute of type ID As shown in the example above the descriptive element UPDATE is very similar to the SELECT element in the previous example note that the REFERENCE element is exactly the same An update query contains an additional lt TAG gt element this element contains the updated version of the item to be updated This element might for example contain an entire Project document the referenced project would then be located and updated with the updated version Deletion of elements lt xml version 1 0 gt C 18 lt QUERY gt lt DELETE ELEMENT_TYPE MASS_SPECTRUM gt lt REFERENCE NAME MASS_SPECTRUM gt lt REF_ATTRIBUTE NAME SAMPLE_TRACKING_ID VALUE B001 gt lt REFERENCE gt lt DELETE gt lt QUERY gt Deleting a Mass Spectrum document for a given Sample Tracking
104. OKYFILLNFVRTKGKYNTNIIKHL Within this general format many different conventions are used If FASTA format is specified as a Databank option you must also specify the correct FASTA format subtype FASTA STANDARD Description line gt NAME ACCESSION NUMBER DATABANK OF ORIGIN DESCRIPTION Example gt IF3 AQUAE 067653 SPT Translation initiation factor IF 3 MSKLKEYRVNROQIRAKECRLIDENGQOQIGIVPIEERALKIAEEKGLDLVETAPOQAKPPVCK IMDYGKFKYELKKKEREARKKOREHOTEVKDIRMKVRIDEHDLOVKLKHMREFLEEGDKV KVWLRFERGRENI YPELGKKLAERI INE LSDIAEVEVQPKKEGNFEMI FVLAPKRKK FASTA NCBI_EXPASY_STANDARD This format comes in two different forms a 2 pipe version and the 4 pipe version shown below The description line of this particular databank format is not shortened in any way Description line gt gi NUMBER DATABANK OF ORIGIN ACCESSION NUMBER LOCUS _OR_NAME DESCRIPTION E 9 Example of 4 pipe version gt gi 3845261 gb AAC71934 1 metal binding protein DHHC dom ain M WCH KMDHHCEWIN SCVGLYNOKYF1 LLNFVRTKGKYNTN Plasmodium falciparum 3D7 KCLCTNPGFLNETFHEFVS DNTTEYDNNVOQMCKKCNLLKI KHL Example of 2 pipe version gt SP PLASM FALC Plasmodium fal M
105. OPSGGGDDFFLVLL PGLOGDESGANPAGCSAQGPHCLSAVPTPAPI Keratin 8 pseudogenes complete sequ 8 Cytokeratin Contains mat LTLAT PP PHAWE PGAAPAQOPRCLIAPOAGF POAAHPGDCPI POREARGLAAALGPRGLLGSGPGVVLY LC PEALCGOTFAKK HLOS KCPLGGCGWTFTTSYKLKR KCEVCEESFPTOAKLGAHOI SCSFPGCSKOYDKACRLKI MCPVEGCGKSFTRAEHLKG RSHF nce gb AL034396 PAGGGRVHRGPDSPAGQVPTRRLLLPRGPQDGGPGRRRE DPVGGDVETAGSGQAAGPVLI SAPGPAAAFAGTVTI HNQ EF LRSDLLLA HDKLRPFGCPARGCGKSFTTVYNLKAH HE PERPYOCAFSGCKKTF I HLRS HSI T HTGERPFLC DFDGCG TVSALFSHN VNETSMSKLLRH HLGTKPFVCPVAGCCARFSARSSLYIH HOLKMHLLT ung 8 ch ESTs DA gene v myc CYK8 an REEAKAG DLLLRFENGV EPA EPAPAPA MKG KRK SKK WKSRCPISSCNKLFTSKHSMKTHMVKRHKVGQDLLAQLEAANSLTPSSELTSQ T AE VSLFSDVPDSTSAALL DTALVNSG LT RAHFREQ HSSSQGORPF HEQENSF ELF HDDDRRF HLODVDT RONDLSD DVASVSSTLAGHLPANNNNSVGQAVDPPS LMATSDPPOSLDTSLFFGTAATGFOOSSLNMDEVSSVSVGPLGSLDSLAMKNSS PEPQAL TPSSKLTVDTDTLTPSSTLCI HGSQKERNLITVTGSSFLV Description line gt ACCESSION FASTA ACCESSION_ONLY ENSVSELLT PAKAEWSVHPNSDFFGOEGETOFGFPNAAGN Example gt AA917165 et at tctag ttaaggactgtagaat ttaat tgt tcgaatacgttctggatat ttcaaaagagt aag taagcacgcaatataatagaga
106. Oto 14 h Maximum Hits to Return 20 Primary Digest Reagent Secondary Digest Reagent Missed Cleavages Trypsin Fixed Modifications Variable Modifications Exclude Masses Validate Results Filter Monoisotopic or Average Monoisotopic Mass Values Peptide Charge 2 Instrument Type Default this databank search Search Engine Type Select the type of search engine you wish to use for Search Engine Type For details of these attributes see Databank search parameters on page 14 5 7 7 Databank Search attributes Mascot search engine Workflow Designer Databank Search Query m Workflow Fragment lon Search rf lt E Workflow Attribute ate LS PR g h Engine Type MASCOT X Database S Taxonomy Peptide Tolerance MS MS Tolerance Estimated Calibration Error 0 005 Da Protein Mass Oto 200000 Da pl Range Oto14 Mir m Peptides to Match 1 Maximum Hits to Return 20 Enzyme Trypsin None Missed Cleavages Fixed Modifications Variable Modifications Yes None Monoisotopic or Average Monoisotopic Mass Values MH 2 Default Search Engine Type Select the type of search engine you wish to use for this databank search Search Engine Type For details of these attributes see Databank search parameters on page 14 5 5 Set the attributes for the search as required
107. PHE 1 49 0 40 0 15 1 00 OOOOOOOoOODoOoOoOoO00 000 ooog ooo0oo0oo0oo0oo0o000n0n 10 10 Using Expression Analysis to compare and analyze sample groups Sort the results by clicking a column heading Click the heading again to reverse the order of the sort To re order the columns click the heading and drag the column to the desired location For each comparison there is a column The cells in these columns when filled completely contain this information e Ratio of Condition A Condition B a condition is sample or group of samples lt Log of that ratio Standard deviation of the log e Probability of upregulation Typical comparison column cell Ratio Probability ot upregulation 2 94 1 08 0 33 1 00 Log of ratio Standard deviation The text is green if the probability of upregulation is 0 95 or more and red if the probability is 0 05 or less A value of 1 00 indicates that the cluster is definitely upregulated a value of 0 00 indicates that the cluster is definitely downregulated If the cluster or protein only appeared in one of the conditions groups then the name of the group that it appeared in is displayed in the cell If the item appeared in neither of the conditions the cell is blank If the cluster or protein only appeared in one of the conditions groups and appeared in every injection for that
108. Projects B o Sample Manager Sample Manager Gel Manager E Display area Somteiner Menage f for the selected tool Tool lt Hide display tray agp arrow for Tool Expression Analysis tray Databank Search AutoMod Analysis Fy Tool tay A O QO 4 scroll Sample Manager BE buttons Status bar Tool tray The tool tray provides links to all the available tools Use the buttons at the bottom of the Tool tray to navigate through the list of tools see Scroll buttons for the tool tray on page 2 4 To hide or display the tool tray click the arrow KI or D on the splitter bar between the tool tray and the Display Area Note Some tools could have been removed from the list using the Add Remove Tools menu see Adding and removing tools on page 2 4 Therefore there might be fewer tools displayed than those shown in ProteinLynx browser on page 2 3 2 3 The following table details the scroll buttons for the tool tray Scroll buttons for the tool tray Button Action Displays the top section of the tool A tray O Scrolls up the list of tools Scrolls down the list of tools Displays the bottom section of the tool 3 tray Adding and removing tools To customize the list of tools shown in the Tools menu and tool tray 1 Click Tools gt Add Remove Tools Add Remove Tools dialog box Ja Add Remove Tools xi v Sample Manage v Gel Manager
109. ProteinLynx Global SERVER Version 2 2 5 User s Guide 71500125602 Revision A Waters CORPORATION Copyright Waters Corporation 2006 All rights reserv Copyright notice 2006 WATERS CORPORATION PRINTED IN THE UNITED STATES OF AMERICA AND IRELAND ALL RIGHTS RESERVED THIS DOCUMENT OR PARTS THEREOF MAY NOT BE REPRODUCED IN ANY FORM WITHOUT THE WRITTEN PERMISSION OF THE PUBLISHER The information in this document is subject to change without notice and should not be construed as a commitment by Waters Corporation Waters Corporation assumes no responsibility for any errors that may appear in this document This document is believed to be complete and accurate at the time of publication In no event shall Waters Corporation be liable for incidental or consequential damages in connection with or arising from its use Waters Corporation 34 Maple Street Milford MA 01757 USA Trademarks Millennium and Waters are registered trademarks of Waters Corporation MassLynx and ProteinLynx Global SERVER are trademarks of Waters Corporation Windows is a registered trademark of Microsoft Corporation IBM and AIX are registered trademarks of International Business Machines Corporation UNIX is a registered trademark of The Open Group Sun and Solaris are registered trademarks of Sun Microsystems Inc Linux is a registered trademark of Linus Torvalds SUSE is a registered trademark of Novell Inc Red Hat is a registered trademar
110. Results are described in Databank search parameters on page 14 5 Consider Modifications You can specify whether modifications should be considered in the matching of spectra against generated peptides If modifications are considered default all the modifications listed in the Modifier Tool are considered where appropriate The check box is selected by default Clear the check box to specify that modifications should not be considered Consider Substitutions You can specify whether single amino acid substitutions should be considered in the matching of spectra against generated peptides If substitutions are considered default all the substitutions listed in the Modifier Tool are considered where appropriate The check box is selected by default Clear the check box to specify that substitutions should not be considered Specify which substitutions to consider in the Substitution Likelihood attribute see Specifying the likelihood of substitutions on page 14 17 Specifying the maximum substitutions and modifications per peptide In the Max Mods Subs per Peptide attribute you must specify a maximum number of modifications and or substitutions to be considered per starting peptide This figure limits the number of residues per peptide that can be modified or substituted at any one time Example Consider the case after digestion that the following starting peptide is generated ACDEFGHILK 10 residues 14 16 Query T
111. S File cell to open the Select File dialog box 2 Choose a previously saved file MS method file such as that created in the previous section Creating an MS method file 3 Click OK Result The MS file is added to the sample list To add an inlet file 1 Double click in the Inlet File cell to open the Inlet Methods dialog box Inlet Methods Cancel 2 Click a previously saved inlet method file 16 7 3 Click OK Result The inlet method file is added to the sample list To run the sample list 1 2 3 4 Click to start the acquisition In the Start Sample List Run dialog box select Acquire Sample Data In the Samples frame specify the samples to run Click OK When the acquisition has finished the raw data can be processed in ProteinLynx Global Server 16 8 Using MS for qualitative proteomics Quick Start Tutorials The following sections cover several common tasks that you might perform using PLGS It is reeommended that you are familiar with the software before attempting these procedures Refer to Chapter 5 Specifying samples vials and plates with Container Manager and all other chapters for details of how to use the software Ensure that PLGS is running on the computer you are using and also on the server if one is being used For information on how to start PLGS see Chapter 5 Installing ProteinLynx Global SERVER Contents Topic Page Creating a project an
112. SCOT ProteinLynx Global SERVER automatically helps you to organize curate your data See also The meanings of identity threshold and homology threshold in relation to the MASCOT search engine are discussed on the Matrix Science website www matrixscience com PMF Automatic data curation rules Search Requirements for Requirements for Auto curation n engine OK assignment Maybe assignment PLGS No MASCOT Yes proteins 95 identity Not provided threshold PMF Fragment lon Automatic data curation rules Search Requirements for Requirements for Auto curation n f i engine OK assignment Maybe assignment PLGS No MASCOT Yes proteins 95 identity Homology threshold threshold B 7 Fragment lon Automatic data curation rules Search Requirements for Requirements for Auto curation iny y engine OK assignment Maybe assignment PLGS Yes if All assigned OK Not applicable Validate Results search parameter set MASCOT Yes proteins 95 identity Homology threshold threshold Electrospray MS Automatic data curation rules Search Requirements for Requirements for i Auto curation n t engine OK assignment Maybe assignment PLGS Yes 95 probability 50 probability MASCOT Yes 95 identity Homology threshold threshold Electrospray H
113. ST match for each spot in the gel Each row includes the gel spot coordinates and similar information to that found in the corresponding workflow results windows see Chapter 6 Viewing results in the Results Browser 9 9 Viewing sample annotation To view the annotation for a sample in any given microtitre plate well or target plate spot click the well or spot icon right click and then click View Sample Information A sample display pane and results window are shown in the desktop area 9 10 Viewing and processing gel data with Gel Manager 1 0 Using Expression Analysis to compare and analyze sample groups Expression Analysis identifies and extracts pairs of labeled masses computes their relative abundance and indicates whether they are upregulated or downregulated Expression Analysis enables you to perform expression profiling experiments Contents Topic Page Getting started with Expression Analysis 10 2 Experiment Analysis Design Manager 10 3 Viewing Expression Results 10 10 Log Plot Viewer 10 18 Expression Data Viewer 10 20 Exporting Switch Lists 10 23 Importing Significant Clusters 10 24 Assess Data Quality viewer 10 25 10 1 Getting started with Expression Analysis The Expression Analysis tool enables you to perform the following tasks with ProteinLynx Global SERVER Take mass spectrum data from samples labeled with different mass tags Identify and extract pairs of labeled masses Co
114. TTD 712 04 BFAPKTTSVRPRKRKA DVAITHLODP KOCASQACWNPDTGYTSPCRR PTPD EVE PTRSTPLPALCWASKD LDWLMEVCEVYKLHRETFYLGQ AAKMEETYPPRKV WLNIYMOMAYLK LAASALFHFSSL ETA ELV SSEKP EST S E 12 Databanks Formats EVWNNLLGKDKLYL DY FDI HOFAYVTDGACT EVLTAOY POATFVOQ KVSGLKWC FKGIAADDMHNIOTHVPYLEWLGKVHSYOLV R EPVAFGSVGFTOYASE DTRVMERHPNLQOPKMRATI RFMATOENVLKTTLOL G LSME DD MKELN A ELLDLC G DLEECVR VMVPFAMS NVDNOFNYVPNN LDV D HLVSF G G OF PWRY DC SENYKRVVVNNMDKTAVKGNM FNVVLKTVSES YFQWDR KPCRLT T EAT MRPAEV NYTN FNT YGPLTA DASA GAMK VYEKS G D EVT GISCLF VSLSPLTPVA RSLEFSYSL REAGSSALKT D ESSORSPVPTGVLTPPP FASTA SRS Description line gt ACCESSION Example gt AAI17165 cttctagttaaggactgtagaataagcacgcaatataatagagagtacgtgggttttata atttaattgttcgaatacgttctggatattatcatacttcttcgttcgttcgttatttct ttcaaaagagttgtaatgaactaaaaacgtataagcaatattcaacttaacaacacaaaa aag FASTA ARABIDOPSIS_GENOME Description line gt ACCESSION ENTRY NAME DESCRIPTION Example gt
115. Viewing res h S oaan EEE seeds sshansessesecvesgusssusseseisede saves sacsaxeesecess 6 2 Re salts DrowsSer suksrisssiodniiessssrnmieoaea dieas Enana ECNE EEr EA Sa E Eiai 6 3 Resnika tees TOD eae ei ei eee 6 4 Bortom aola eai E a eve hoops Una ee RaernET TE 6 5 viii Table of Contents Gpectrum viewer toolbar ernaria anaa ET 6 6 Results browser navigator tre sosirii ki ek a aa ii 6 7 PIOA E a hehe a aaa eis 6 7 AA e gent EETA E EEE A A EE E E A EE A A E E EE e A 6 9 Selecting items in the navigator tree cccccccscccecccecceccceccceccescesccsecceseneeeess 6 9 Le Eas eee eee ae een ee een eee eee een ree mt E 6 11 Prot in Ee norec ENA 6 12 Pep dt Oe eies Ea oE Nai 6 13 Controlling thecolumns in the tables oss cccasc cscs cccasexiassaeassansarsaiaarserarsscansasians 6 14 Selecting proteins and ESTs from the table cccceecccccecceeeeeesentaeees 6 15 Selecting peptides from the table ccccccccccccccccecceccceccccecceeccesceseeseeenss 6 15 Resubmilthiag the gear honan S A 6 15 ae a a E E P E E E inset E E EE E 6 16 Printu he resili ee ee ee eee ce ee eee 6 16 Spectrum Viewer for MS data ccecccescscceseessccceccsecceseeeceseeeeeeeeeeeeeeeeeneees 6 16 Viewing raw dola nianna aa 6 18 Changing the sasis VIEN sarosane a ES 6 20 Viewing the fragment ion display sssosssosssoososssosssossesosrossessreosresserereeseee 6 20 Spectrum Viewer for MSMS data icccccccsccaistdeisteinavedsaccinarteisaacinateinaiecnnneeans 6 21 Displaying
116. W spectrum data is processed and whether certain attributes for example smoothing are considered To open the Data Preparation tool and create a new template 1 Click the Data Preparation icon on the tool tray The Data Preparation window opens Nothing is displayed in the main window 2 Click on the toolbar A panel appears from which you can select an acquisition type for the template Data Preparation tool selecting a type of acquisition Data Preparation Create New Processing Parameters Select Acquistion Type Maldi MS Maldi PSD MX Maldi QTof MS Maldi QTof MSMS Electrospray DDA Electrospray MS Electrospray High Low O Q 8 2 Creating custom processing parameters 3 Select the type of acquisition that generated the raw data and then click O A data preparation template is displayed on the Desktop panel and an Editor panel is displayed in the left hand panel The next graphic shows a new MALDI MS processing template Data Preparation tool display Data Preparation Processing Parameters m Maldi MS Atinbute Mass Accuracy Attribute Set Processing Parameters crea E D MALDI MS Noise Reduction Attribute Panel L D maLo ms Enter a title for the data preparation template D MaDi and Centroiding L C maLo ms Title Processing Parameters created on Fri Jan 03 2003 at 11 02 56 Data Preparation Template Editor Panel Desktop Panel By d
117. Workflow Template XML File dialog box and then click Open Click the new workflow template that has been added to the navigator tree and then right click Click Start Workflow to start the process A prompt for a workflow title is displayed Click OK to start the process To display the results click the new workflow template 5 20 Specifying samples vials and plates with Container Manager Adding processing parameters templates So far all the processing has been done using the default processing parameters However different Processing Parameter Template files can be attached to the Raw Data Spectrum Node of the navigator tree Once added all the templates that are part of the project are displayed under the Processing Parameters Templates node See also Processing Parameter Template files are produced with the Data Preparation tool see Creating custom processing parameters on page 8 1 for details To add processing parameter template files 1 In an unprocessed Raw Data Spectrum Node for a well click the Processing Parameters Template and then right click 2 Click Change Processing Parameters 3 In the drop down list click either Choose new processing parameters template from file or one of the Processing Templates Rule The Processing Parameters Templates that appear in the drop down list are those that are already part of the project and are listed under the Processing Parameters Templates node in
118. a ProteinLynx Browser File Edit View Windows Options Tools amp 8 fF S amp S B F Projects testt DOr Container Manager m test_pepgrab i ubmi ass ubmitte arge perimel ass el Da el el ppm K ES Workflow Results 4 peptides Submitted M Submitted Ch timental Mass __mvW__ Delta Da Delta Delta ppm Prot uth 471 811 2 941 606 471 811 2 941 606 941 0 04 0 04 42 863 35 81 Ci noe yt 507 841 2 1013 666 ith 514 344 1 613 336 amp 2 is max Container Manager There can be short delay between selecting the peptide in the tree and rendering the data for display 6 18 Viewing results in the Results Browser In the graph the coloring is Black a high density of data Red a low density of data To zoom into the raw data use the zoom function which is described in Spectrum Viewer options on page 6 24 As you zoom in to levels nearing that of the data dots represent the actual mass intensity points The graph color changes to red which shows that the data is not dense Error messages There are several error messages which could be displayed if there are problems retrieving the data these are detailed in the following table Viewing raw data error messages Error Message Error connecting to processor please start the processor Suggested Course of Action The raw data viewer needs the processor to be running restart the processor For
119. a srera a r e o ai a ia 5 13 Selecting more than one well or spot cccesseseeeesceeceseeseeceseeeseseseeseneees 5 14 Processing raw data cccssecesssscadesssensesssecscsusseaseseseusseo0sceesescsceascaetseiscessendsceesesase 5 17 Workflow and spectrum icons in the navigator tree ssssessssssssssesessssesss 5 18 Vienne the nines oper rU opsir enson ESSER 5 19 Re searching processed data ccccccccccsccsceccccccceccecececcseccscecsccsscesseesscsesecees 5 20 Adding processing parameters templates cccccccssccssccsccssscesscssessseeees 5 21 Exporting and importing mass spectra essesessessessssessssssssssesssesssssssessseoss 5 22 Ezportine mass speca oipe casenenat dieses Sei ein iaewcc alae aie 5 22 Tapers ASS SSCS aara SSE S 5 22 Working with plates ccissessssscessicsiessestacesteisescdess a eaaa a a aanas aaa 5 23 Merging MSMS spectra and results sroscarianesier dadaa 5 24 Customizing ihe pate VIEW pneri ET 5 25 Simplifying peaks with SuperTrack sesssesssesssossessssossossoccoeosoecoeecseesoeeoseeso 5 26 Exporting SuperTrack results as XML cccccceeeeeeeeceeeeeeeeeeeeseeeeeeees 5 28 Interfacing with Massliy x seisis toese searen iorns EE ERa rR 5 29 Exporting a sample hst to Mass Lynx srcrrsrerenirasinienrarn niin ni 5 29 ACOE a T 5 31 Troubleshooting failed client server workflows cccccccccccceecceccescees 5 33 6 Viewing results in the Results Browser ccccccccccscccsrsssssnsssssscesees 6 1
120. agcaagtc ggagcactaa agcagtttgg caaatttaaa 180 E 5 Genbank flat file format The Genbank format is specified by NCBI Example LOCUS AAC71934 101 aa linear INV 16 APR 2002 DEFINITION metal binding protein DHHC domain Plasmodium falciparum 3D7 ACCESSION AAC71934 VERSION AAC71934 1 GI 3845261 DBSOURCE accession AE001414 1 KEYWORDS SOURCE Plasmodium falciparum 3D7 ORGANISM Plasmodium falciparum 3D7 Eukaryota Alveolata Apicomplexa Haemosporida Plasmodium REFERENCE 1 residues 1 to 101 AUTHORS Gardner M J Tettelin H Carucci D J Cummings L M Aravind L Koonin E V Shallom S Mason T Yu K Fujii C Pederson J Shen K Jing J Aston C Lai Z Schwartz D C Pertea M Salzberg S Zhou L Sutton G G Clayton R White O Smith H O Fraser C M Adams M D Venter J C and Hoffman S L TITLE Chromosome 2 sequence of the human malaria parasite Plasmodium falciparum JOURNAL Science 282 5391 1126 1132 1998 MEDLINE 99021743 PUBMED 9804551 E 6 Databanks Formats REMARK Erratum published erratum appears in Science 1998 Dec 4 282 5395 1827 REFERENCE 2 residues 1 to 101 AUTHORS Gardner M J TITLE Direct Submission JOURNAL Submitted 02 NOV 1998 The Institute for Genomic Research 9712 Medical Center Drive Rockville MD 20
121. age 5 25 To view the results do one of the following actions In the navigator tree click the name of the workflow Inthe Results Summary table click the relevant row For details about the results display see Chapter 6 Viewing results in the Results Browser Workflow and spectrum icons in the navigator tree As the raw data is processed the icons displayed in the navigator tree change to indicate the progress of the workflow Navigator tree processing icons Icon Description No raw data is attached to the mass spectrum node AA Unprocessed data is attached to the mass spectrum node Processed data is attached to the mass spectrum node Rule Applies to data processed in the browser or imported as an XML file attached to the mass spectrum node Data that has been processed with SuperTrack is attached to the mass spectrum node A workflow template is attached but not processed l i Processed data that has been successfully lockmass corrected is 4 Processing of the workflow template has failed See Troubleshooting failed client server workflows on page 5 33 Processing of the workflow template is in progress failed Processing of the workflow template is complete Click to view results Browser displaying processed data on page 5 19 a Processing of the workflow template is complete but has partially 5 18 Specifying samples vials and plat
122. al time databank searching To enable real time searching of databanks there are a number of essential steps to take before the system will operate correctly To enable real time searching 1 Ensure you have launched the Real Time Databank Searching application Launching the Real Time Databank Searching application on page 15 2 Set up the acquisition by see Setting up a real time databank searching acquisition on page 15 8 Creating a conventional MassLynx DDA acquisition method Running the ProteinLynx databank search engine microkernel Enable real time processing for processing raw data and query submission Edit raw data processing parameters according to your requirements see Processing parameters on page 15 4 Edit the databank searching parameters including setting the appropriate databank see Searching parameters on page 15 5 Start a MassLynx acquisition using the appropriate DDA method Display the databank results Real Time Status during the acquisition see Real time status on page 15 7 Launching the Real Time Databank Searching application To launch the ProteinLynx Real Time Databank Searching application 1 In MassLynx click the Instrument tab and then click the MS Method icon 15 2 Real Time Databank Searching MS Method editor launch XA MassLynx ProfileLynx CHI_calculation SPL File View Run Help 2 OREH J oo G Shortcut yc
123. altering the behavior of these modules Specifying additional formats in which spectra can be saved after processing Altering the modules PlugIns that handle archiving and retrieval of ProteinLynx project data Contents Topic Page ProteinLynx browser 2 2 Changing preferences 2 5 Setting Automation Setup parameters 2 18 2 1 ProteinLynx browser The user interface for PLGS is the ProteinLynx browser which provides access to various PlugIn tools in the ProteinLynx suite see Figure titled ProteinLynx browser on page 2 3 The ProteinLynx browser enables you to View and edit global preferences View and edit automation set up parameters e Change between tools Manage the desktop which is shared by most of the tools The content of the toolbar and menus varies depending on which tool is selected The Preferences button is the only button common to all toolbars The following commands are common throughout the software from the Menu Bar File gt Exit Options gt Preferences see Changing preferences on page 2 5 e Options gt Automation Setup see Setting Automation Setup parameters on page 2 18 lt Tools gt Add Remove Tools see Adding and removing tools on page 2 4 2 2 Setting up ProteinLynx Global SERVER ProteinLynx browser Title bar Menu bar Tool title panel 15 x File Edit View Windows Options Tools Toolbar i amp
124. alues to their default assignments Copy an image of the protein or peptide tree to the clipboard io Bottom toolbar A toolbar at the bottom of the results browser enables you to quickly open windows and switch between views Results browser bottom toolbar buttons Button Description View the Protein Results panel View the Peptide Results panel View the MS Spectrum panel View the MSMS Spectrum panel Show the BLAST Basic Local Alignment Search Tool results see BLAST results on page 14 26 for further details Show a web page containing the original Mascot results Available if the search was performed against Mascot E e m m oo G Results browser 6 5 Results browser bottom toolbar buttons Continued Button Description Opens the Protein Workpad see Protein Workpad on page 6 27 E E Open the PepGrab Parameters dialog box Available if the databank used is indexed for running PepGrab see PepGrab on page 6 11 for details B Prints the results of the workflow Spectrum viewer toolbar A toolbar to the right of the spectrum viewers enables you to switch between spectrum views and to copy spectrum data Results browser Spectrum viewer toolbar Button mE Description View the MS spectrum lt View the raw data View the expected fragment ion masses Show the retention time
125. ameters see Defining templates for searching with Workflow Designer on page 7 1 and Creating custom processing parameters on page 8 1 5 2 Specifying samples vials and plates with Container Manager Importing and viewing PLGS sample lists Sample lists can be used to organize the samples you want to work with You can create a list of samples to be processed using ProteinLynx Global SERVER and then import that list into PLGS Rule PLGS sample lists tab or comma delimited text files are different from MassLynx sample lists Sample lists are one way of organizing the samples you want to work with you might find them more convenient than identifying samples by vial microtitre plate or target plate Importing PLGS sample lists Requirements Certain requirements apply to sample lists that you intend to import For details see Sample list requirements on page 5 4 To import a sample list 1 In the navigator tree click Sample Lists and then right click 2 Click Import Sample List 3 Inthe Sample List Chooser dialog box browse to the sample list file you wish to import and then click Open 4 Type a title for the sample list This title is the name that is displayed within ProteinLynx Global SERVER Results The imported sample list is added to the navigator tree under Sample Lists The samples specified in the list are added under a node that bears the title you specified when you imported the list The
126. amples v Inject Volume Process Sample ID 5 000 PeptideAuto2 1047040344 operate o Ready 6 Q Time Pressure 18 95 min 1656 psi a MS Tune MS Method Inlet Method Flow 6 000 l min A 92 0 8 0 Q Flow Pressure J 1 120 pl min 16 psi Not Scanning ii Only Error Shutdown Enabled x A 23 PLGS data processing consists of two major steps Processing MS data lock mass correcting and generating lists of precursor mass and charge state Processing the MSMS data again lock mass correcting and deisotoping data When the sample data has been processed and searched against the database the display in PLGS can be updated To update the display for the current project in PLGS click File gt Update PLGS with acquired data File Edit View Windows Options Tools B BELa DTO K Giova Server Example Data Container Manager Global Server Example Data 888 vials Microtitre Plates 12 o OK Accession Entry E sample plate 1 gt nilie Serum albumin precursor P02768 _ ALBU_HUMAN Serum albumin pre Sample Manager Data Preparation 7 P02787 TRFE_HUMAN Serotransferrin pre gt nille Serotransferrin precursor Siderophil P01922 HBA HUMAN sg men x gt wiilce Hemoglobin alpha chain S pemanroled gt nille Beta globin gene from a thalassemi miz z Peakmw Peptide mw 1226 6039 1225 5961 1225 597 2490 2952 2489 2874 2489 277 1149 6178 1148 6100 1148 607 m Results
127. ance from the tallest peak in an isotope cluster to the end of the cluster in Da Figure titled Peak Detection tab on page 15 11 Extraction window Once a peak has been selected by the peak detection window a section of the mass scale around the peak is taken for deisotoping An ideal setting for this value is half the overall peak cluster size Figure titled Peak Detection tab on page 15 11 Exclude tab Function 1 Survey Scan Adducts Collision Eneray LockSpray Variable Flow Acquisition MS Survey MS MS Peak Detection Exclude Include Dynamic Peak Exclusion IV Enable real time exclusion of masses from MS MS Acquire once then always exclude for the rest of the acquisition Acquire and then exclude for 10 seconds Exclude peaks within 100 mDa of entries on the exclude list 10 e Fixed Peak Exclusion Ee I Exclude from range I Exclude from file fad Exclude Mass Retention Time 15 12 Real Time Databank Searching Exclude window The exclude window on the Exclude tab Figure titled Exclude tab on page 15 12 can then be set to 100 mDa or lower if desired Other DDA experiment settings Other settings are comparable to a normal DDA experiment 15 13 Advanced options The following are advanced options in the ProteinLynx Real Time Databank Searching application e Real time data processing Remote searching Diagnostics Data processing To a
128. ank attributes You can change the values for the following attributes Databank attributes Attribute Description Name Contains the name of the family of databanks This name appears in the list of databanks in the Databank Search tool and other search tools This field is compulsory and must be set when a new databank is created After the databank has been created and saved this field cannot be changed Type Select from the list of supported databank types Default Protein Format The format of the sequences in the databank flat file Select from the list of supported formats It is important that the correct format is selected so that the databank can be processed correctly and that search results can be displayed in a meaningful way 13 4 Organizing databanks with the Databank Admin tool Databank attributes Continued Attribute FASTA Format Description One of the most widely used formats for specifying sequence information is FASTA format In its most general form FASTA format comprises a one line description beginning with a gt symbol followed by multiple lines containing the sequence of amino acid identifiers Within this general format there are many format subtypes used by different organizations If the format of the databank is FASTA use this field to specify the particular FASTA convention which is used From the list of supported FASTA formats select whichever
129. ard To uninstall PLGS using the graphical user interface GUI 1 Navigate to the uninstall folder 1 8 Installing ProteinLynx Global SERVER _uninstall folder File Edit View Go Bookmarks Help I gt 1 40 A Back Forward Up Stop Reload Home Location ust local PLGS2 2 _uninst 00 View as Icons Y uninstall dat uninstall bin uninstall jar 17 6 K 2 3 7 MB 2 Double click uninstall bin 3 Follow the instructions in the Uninstaller Wizard Installing PLGS on Linux PLGS can be installed from a command prompt or by using the graphical user interface GUI Linux will automatically detect when you load the installation CD Requirements If you are installing on SUSE Linux you must ensure that the IBM C Runtime Libraries are installed and that the Java JIT compiler is turned off For further assistance refer to the ProteinLynx Global SERVER Release Notes To install PLGS from a command prompt 1 Open a terminal window and navigate to the installation directory using the command cd usr local 1 9 1 10 Running InstallShield v root localhost ust local File Edit View Terminal Go Help root localhost root cd usr local root localhost local ls 1 total 184368 drwxr xr x 6 root root 4096 Jun 17 18 56 Acrobat5 2 root root 4096 Jan 24 2003 bin 2 root root 4096 Jan 24 2003 etc 2 root root 4096 Jan 24 2003 games 2 root root 4096 Jan 24 2003 include 2
130. as been PlugInImp declared in the Class Path field the list of classes found in the plugin that implement the interface PlugInImp are displayed This is for your information only and is there only to confirm that the plugin does implement this class Properties You can add remove or modify any properties required by the PlugIn for example the working directory of the PlugIn To add or modify a property click Add or Modify Type the values in the Add Modify dialog box that opens To remove a property select the property and then click Remove Export Selected Select this to export selected results from a Results from Container container directly to the PlugIn Default Cleared Save Projects from Select this to execute the PlugIn whenever Browser and projects are updated by the browser or PeptideAuto PeptideAuto Default Selected 3 In the PlugIn Selector dialog box click OK Result For an Import PlugIn the new PlugIn replaces the previous PlugIn For an Export PlugIn the new PlugIn is added to the list 4 On the PlugIns tab click OK Requirement For the PlugIn to work the ProteinLynx Browser must be restarted Modifying an Export Plugin You can modify the details of any Export PlugIn including the supplied PlugIn 2 27 To modify an Export Plugin 1 On the PlugIns tab select the PlugIn from the list 2 Click Modify The PlugIn Selector dialog box opens Figure titled
131. asted or dragged and dropped into the text area The sequences must be in fastA format Tip fastA format sequences can be added by dragging and dropping ESTs from the navigator tree or protein table in a ProteinLynx search results frame 14 18 Query Tools De Novo Sequencing tool De Novo sequencing enables you to determine the primary sequence of a peptide directly from its MSMS data This is achieved by analyzing the mass differences between the peptide fragment ions This tool facilitates the characterization of peptides whose protein or EST has not yet been entered into a databank and generates sequences that can be subsequently used in a BLAST search You can use the De Novo Sequencing tool to search data from any instrument that can generate fragmentation spectra Electrospray Q Tof Maldi PSD and Maldi Q Tof This type of analysis is primarily used as the third step in a workflow to sequence MSMS data not matched by a Databank or AutoMod query De Novo sequencing can also be carried out as a one off query where all the available fragmentation data is sequenced Note Adding a De Novo query to a workflow differs only slightly from carrying out an individual search and so the following section contains information relevant to both types of experiment To open the De Novo Sequencing query tool click the De Novo Sequencing icon ae in the tool tray The De Novo Sequencing Parameters table opens in the Editor Panel of the br
132. ata Euteleostomi Actinopterygii Neopterygii Teleostei Ostariophysi Siluriformes Ictaluridae 1 1 187 Ictalurus Liu Z Tan G Li P Dunham R Transcribed dinucleotide microsatellites and their associated genes from channel catfish Ictalurus punctatus Unpublished E 3 E 4 XX DR UNILIB 1529 1529 XX Ce Other ESTs IpTRO40r EC Contact Liu Zed CC Fish Molecular Genetics and CC Auburn University CC 203 Swingle Hall 36849 USA EC Tel 334 8 AQ 54 CE Fax 334 8 9208 CC Email zliu acesag auburn edu Ce Seq primer M13 forword CC High quality sequence stop 187 XX FH Key FH FT source FT FT FT FT FT FT FI library FT FT XX SQ Sequence 187 gggggaaaaa aaccaaacaa acaattacag caggcgcgaa gcaccgatat cggattagtg BP 60 Biotechnology Department of Fisheries Auburn AL Location Qualifiers Lerla db_xref taxon 7998 P1529 db_xref UNILIB sex female organism Ictalurus punctatus strain Kansas clone IpTRO40 clone_lib Chan nel catfish pituitary tissue_type pi tuitary dev_stage adult 58 A 36 C 50 G 43 T O other cgtgaacgat accttgagct agtcggtggg acagtcggct aatgctagct ttgcgattaa Databanks Formats 120 cgtg tcattc tatgcagtt gagcttt cg
133. ata rs E te ected eee es ee ee ee 15 14 Rents Sea KONING sus S R iene 15 14 Dieplaying diagnostte addicts tice edie 15 15 16 Using MSE for qualitative proteomics ssiisssccisccccacdisssssvesccrascsscosseaes 16 1 What is MSE sssssssssssissessosspsstsssistsstessrssnesiitdsriknieissttiirtcrisaibsi tidintasbadtridtsitisid 16 2 Creating an MS method file s ssessessessesssssessessossessessersroscssesseseoseossessesseseeses 16 3 Running an MSE experiment esssesssssesessesssessesorseesesseossessesceseoseossoseoseoseesesse 16 7 Necessary sample list Tell 8 ios ccc esiescenenaieunsnereninninsecenectenecens 16 7 A Qu ick Start Tutorials vcscsscaicisssasecssssnatetsvereesiaceleniiaentennaaone A 1 Creating a project and processing acquired data files ssscssseeseees A 2 ER A A E E O E E E E E E E E E E A T A 2 S ttimne the target plate rnern rE EKE IRSE Ns A 2 MALDI test procedure sicsiiacieseseesevesvessecsedasesdodescsccawisedsasedsdavedseveccwedecsacesssaressere A 5 Setting the target plate s ccvusianrciexestaanadvaacansaeddvainsanmavoniavensundowndoeanenion nuevas A 5 Table of Contents xv Setting processing Parameters osrin nonn rn E TEE A 6 Creatine a wOrkHOW aorari nan A EA A T Attaching the data processing parameters cccceseeseeseseesesseereeenterseeees A 8 Atacmag the work ow TG cansei R A 9 Exporting the sample list to Mase Lye cccsvessesccscssccsssvarsevarcassccssecansevarsavens A 9 FOTO E bt Ramtec een Cree E E E
134. ated by the theoretical digest However if search times are critical you need to consider carefully the use of this attribute Example If a single variable modification is applied a peptide containing three amino acids that bond with the modifier will generate eight variations in Fragment Ion searches and four in PMFs To specify a modification that should always be applied to peptides produced by the digests click the desired reagent in the list To select multiple reagents in the list use Shift click to select consecutive reagents or Ctrl click to select non consecutive reagents Exclude Masses Rule This attribute applies only to PLGS PMF searches This attribute specifies masses that are to be excluded from a search These excluded masses could include masses of known matrix impurities contaminants or lockmass peaks If the specified masses appear in the submitted spectra to within the supplied peptide tolerance these masses are suppressed when performing the search The masses are not actually excluded but their influence is suppressed as it is assumed that the peaks belong to a contaminant Therefore while excluded masses can sometimes be matched the influence that these peaks contribute to the final score is suppressed In the text box type the masses that are to be excluded separated by a space or return MALDI only 14 11 Masses selected for exclusion are usually theoretical masses which can differ from masse
135. ates and microtitre plates To display the Plate menu click a well or drag across a number of wells and then right click Plate menu Select All View Results Merge Results Set Sample Set Attached Templates View Sample Information View Attached Templates gt gt Import Mass Spectrum Set Raw Data File Process Plate Settings You can use the following menu options Plate pop up menu options Option Select All Description Selects all the wells on the plate View Results Opens the results browser see Viewing results on page 6 2 Merge Results View Sample Information View Attached Templates See Merging MSMS spectra and results on page 5 24 Displays sample information on the right hand panel Select to display either a workflow template or processing template 5 23 Plate pop up menu options Continued Option Description Set Sample Described in Setting a sample on page 5 11 Set Attached Set the processing and workflow templates Each option Templates will open a dialog box in which previously saved templates can be selected Import Mass Spectrum This option is the same as described in Importing mass spectra on page 5 22 Set Raw Data File This option is the same as described in Attaching raw data on page 5 13 Process Process raw data or latest data Plate Settings See Customizing the plate view on
136. ator tree click Target Plates and then right click Click New Target Plate New Container dialog box New Container Container Type Microtitre plate Target plate Vial 8 X 12 TARGET PLATE v OK Cancel In the Barcode text box type a title or identifying number If required select a format for the plate Click OK In the navigator tree expand the Target Plates node and then click the new plate Result Two new displays open The Plate Viewer below the navigator tree displays a graphic of a target plate 5 9 New target plate display Container Manager CSF_225Processing BiG vials EP Microtitre Plates EA Target Plates o Barcode Sample Lists Processing Parameters Templates Workflow Templates New Target Plate 36 36 36 36 3636 36 36 3636 36 36 363636363636 3E 3E 3E IE 3b 38 Container Manager 5 10 Specifying samples vials and plates with Container Manager Setting a sample See also For details about how to create samples see Annotating and tracking samples with Sample Manager on page 4 1 If a vial microtitre plate or target plate is being used the vial or plate must be associated with a PLGS sample manually If a sample list was imported each data file whether raw or processed is already associated with a sample To set the sample 1 Open the Select a Sample dialog box following the instructions in the following table Setting samples
137. ave button Result The new reagent is added to the list in black text 12 5 Deleting custom modifier reagents To delete a custom modifier reagent click the reagent in the list and then either e Click File gt Delete Click the Delete button T 12 6 Managing modifier and digest reagents Getting started with the Digest Reagent tool The Digest Reagent Tool enables you to manage all digest reagents used in the ProteinLynx system You can View the properties of the large number of digest reagents that are supplied with ProteinLynx Define your own digest reagents which are immediately available to the full suite of ProteinLynx browser tools Va To open the Digest Reagent Tool click the Digest Reagent Tool icon on the tool tray A list of digest reagents is displayed Supplied reagents are shown in gray text custom user defined reagents are shown in black text 12 7 Viewing existing digest reagents To view the properties of a reagent click a reagent in the list The attributes and values are displayed in the panel below the list Digest Reagent Tool Digest Reagent Tool Digest reagents Chymotrypsin Slymotrypsin EndoLys C S Aureus pH 8 View Digest Reagent Name Trypsin See New digest reagent attributes on page 12 9 for details of the attributes and values Rule The values of supplied digest reagents gray text cannot be edited 12 8 Managing modifier and dig
138. aving custom digest reagents To save the new or edited digest reagent click the Save button B Result The new reagent is added to the list in black text Deleting custom digest reagents To delete a custom digest reagent select the reagent in the list and then either Click File gt Delete e Click the Delete button T 12 10 Managing modifier and digest reagents 1 3 Organizing databanks with the Databank Admin tool Contents Topic Page Getting started with the Databank Admin tool 13 2 Adding databanks 13 3 Editing databanks 13 11 Removing and deleting databanks 13 13 Connecting to a search engine 13 17 13 1 Getting started with the Databank Admin tool Databanks are flat files that contain information regarding sequences of nucleotides or amino acids These files are used by the Databank Search and the BLAST Searching tools The Databank Admin Tool Enables you to organize databanks and choose databank properties Regulates any automatic downloads and updates Generates auxiliary files that are needed by the other tools when performing searches Enables you to view the databanks that reside on the currently connected search engine O To open the Databank Admin Tool click the Databank Admin Tool icon in the tool tray Tips A search engine must be specified see Changing preferences on page 2 5 for the Databank Admin options to be available If there are no databanks displayed
139. ay click the icon for one of the tools that requires a project Sample Manager Gel Manager Container Manager or Expression Analysis If the project is not currently displayed switch to the project you wish to delete see To open a project on page 3 5 for details Click the name of the current project in the navigator tree Click Edit gt Delete If you are sure you want to delete the project click Yes Result The project is deleted and is no longer available in the ProteinLynx browser Processed data is deleted but the original raw data is not 3 6 Creating importing and managing projects Annotating and tracking samples with Sample Manager Sample Manager enables the full annotation and tracking of all the samples used in a ProteinLynx project Contents Topic Page Getting started with Sample Manager 4 2 Sample editor 4 3 Getting started with Sample Manager The Sample Manager enables you to fully annotate all the samples used in a ProteinLynx project Individual samples can be named and associated with hyperlinks allowing clear sample tracking throughout the whole ProteinLynx system Also individual samples can be mixed to produce processed samples which include full details of their origin When you set a sample in Container Manager see What is Container Manager on page 5 2 you choose from the samples that you added to Sample Manager The samples specified and configured in Sample Manager are als
140. ays after which an automatic interim update will be undertaken The details of this attribute are the same as for Download Renew Period Keep Archives Rule This option is only available if one or both of the Periodically Download or Periodically Update attributes have been set to True To keep archived databanks set this field to True These archives can be restored at a later date For details of archives see Keeping archived copies of a databank on page 13 15 Processing Start Time and Processing End Time Format HH MM 24 hour clock Some of the processing steps such as automatic download of large databank files and making blastable when applied to large files can take time to perform During this processing period the databank might become temporarily unavailable to other search tools For this reason it can be preferable to schedule processing to take place only at times when the databanks are unlikely to be needed by other tools The Processing Start Time specifies the time after which all such automatic processing will be scheduled The Processing End Time specifies the time after which no further processing will be scheduled It is important to specify a time period during which the machine is on If there is no preferred processing time set to 00 01 and 23 59 13 10 Organizing databanks with the Databank Admin tool Editing databanks You can only edit databanks that reside on th
141. cesnisesasesearigaczecnvaessessvessacsesiaves saree KETSE ENNERT B 8 Electrospray Hit WLOW roroa B 8 xvi Table of Contents C Implementing a plugin for ProteinLynx Global SERVER C 1 An introduction to the PLGS plugin u cc ecccccccceccceccceccecccecccecceeccecees C 2 Plgsin architecture scccack ces seeeceskscevceseasiccees cedssieees A EN ERE C 3 Use case the PLGS FileSystemPlugIn sesssssssesssesssessosssosssosssossossssoseosseso C 5 XML communication with the plugin implementation ccccceeeees C 6 Adding a plugin to the PLGS application ccccccccceccceccecccceccsccceeees C 7 An example Executable plugin cccccccccecccecceecceecceeceeccececsecssceeseeseceese C 11 An example Java plugin ccccccccccccccccccccecccecccecceccccecceecceccceecsecessecseeseeeess C 13 Basic plugin Specific Queries essssssssssssssosssosssessosssosssoeseosseessesoecoseessesoeees C 16 eleccion Or Sleme npon ied ine ees C 16 Selecting a Project document for a given Project ID eceeeeeeeees C 16 Update or Fos va nL eee rene tere ete RtepeR S C 17 Updating a Project document for a given Project_ID ccceeeeeeneeee C 18 D leton of elemenis suiuineas C 18 Deleting a Mass Spectrum document for a given Sample Tracking ID C 18 Tosertion or BACON erana EE C 19 Inserting a Workflow document and updating the associated Project CONE airain E A C 19 Query tag definitions in the Pr
142. cess Method Subtract Description Mass Measure Survey and MSMS Apply the same MassLynx mass measure algorithm to both the survey scan data and the MSMS scan data Mass Measure Survey MaxEnt Lite MSMS Apply the MassLynx mass measure algorithm to the survey data and perform MaxEnt Lite deconvolution to the MSMS data Select the box to enable background subtraction of the raw data and adjust the settings according to your requirements Smooth Select the box to perform Savitsky Golay smoothing of the data Adjust the smoothing parameters according to your requirements Peak Centering Adjust the parameters according to your requirements MaxEnt Lite MaxEnt Lite will produce a singly charged deisotoped spectrum for interpretation by the search engine Type the molecular mass range of this spectrum the maximum charge expected in the data and a threshold setting For the threshold setting type a negative value for relative percent thresholding or a positive value for absolute thresholding Data below the threshold will not be considered by MaxEnt Lite Searching parameters To view or edit the Searching Parameters click the Databank Searching icon in the tool tray The Searching Parameters view is displayed 15 5 Searching Parameters page ProteinLynx Real Time Database Searching Ox File RealTime Settings Help Searching Parameters r Searching r Peptide
143. ch Engine tab 2 5 search engines adding 2 6 connecting to 13 17 Mascot 7 6 modifying 2 7 PLGS 7 6 removing 2 8 type 14 5 search method AutoMod Analysis 7 2 BLAST Searching 7 2 Databank Search Query 7 2 De Novo Sequencing 7 2 search parameters databank 14 5 for BLAST algorithm 14 24 search type Fragment Ion Search 7 2 PMF Peptide Mass Fingerprinting 7 2 PMF Fragment Ion Search 7 2 Index 16 searching methods 7 2 parameters 15 5 strategy 7 2 searching data from a sample list 5 7 secondary digest reagent 14 10 14 16 14 21 secondary internal lock mass 8 7 Secondary Internal Lock Mass attribute 8 7 Select a Colour dialog box 2 14 2 15 Select Files dialog box 5 14 5 15 Select Processing Parameters dialog box A 9 A 19 Select start time attribute 8 16 Select stop time attribute 8 16 Select time range attribute 8 16 selecting data Expression experiment 10 7 EST 6 15 EST sequences for search 14 18 grouping method Expression experiment 10 5 peptides 6 15 protein sequences for search 14 18 proteins 6 15 URL 14 5 selecting calibration type 8 6 sequencing De Novo parameters 14 21 server starting PLGS 1 19 services installing 1 4 Set Raw Data 5 138 5 15 setting processing parameters A 6 samples 5 11 showing axis labels 9 9 diagnostics 15 15 significant clusters import 10 24 simulated digest 6 33 running 6 29 Smooth parameter 15 5 Smoothing Iterations attribute 8 10 Smoothing Type attribute 8 10 smoothing
144. ck OK The wells change color A 5 Setting processing parameters To set processing parameters 1 2 ee pE 8 Click Data Preparation Click File gt New Select Maldi MS and then click O Result A new Processing Parameters template is opened see MALDI Q Tof MSMS on page 8 5 Name the Processing Parameters template MALDIPP In the Mass Accuracy attributes set the Calibration Type to External Set the External Lock Mass as 2465 1989 Da ACTH Enter values for the Noise Reduction attributes as shown below Noise Reduction attributes Data Preparation MALDI MS Attribute Background Subtract Type Normal Background Threshold 10 Background Polynomial 15 Perform Smoothing Yes Smoothing Type Savitzky Golay Combine Options All Scans to Combine 1500 Da Intensity Range 2to 100 4 Background Polynomial Enter the order of the polynomial with which to fit the background A value of 0 corresponds to a flat threshold and 1 is a sloping straight line For typica data a value of around 5 will be sufficient Background Pohmomial 15 Enter values for the Deisotoping and Centroiding attributes as shown below A 6 Quick Start Tutorials Deisotoping and Centroiding attributes Data Preparation MALDI MS Attribute Value Perform Deisotoping Yes Deisotoping Type Medium Iterations 30 Threshold Centroid Top Minim
145. ckage has its own start up procedure See also Additional platform specific information on installation and configuration issues can be found in the ProteinLynx Global SERVER 2 2 5 Release Notes Contents Topic Page Typical client server installation 1 2 Installing PLGS on Windows 1 3 Installing PLGS on Linux 1 7 Installing PLGS on UNIX 1 15 Restoring old databanks 1 23 Setting the number of processors 1 24 1 1 Typical client server installation The following graphic shows how ProteinLynx Global SERVER is typically used in a client server environment ProteinLynx Global SERVER in a client server environment MassLynx PC with ProteinLynx ty w ps XML Results Returned XML Results Returned A ex we cS Database Server MassLynx PC with ProteinLynx 1 2 Installing ProteinLynx Global SERVER Installing PLGS on Windows This section describes the steps to install and run PLGS on Windows on a single PC or in a client server environment However if you have a previous version of PLGS already installed on your PC you must back up the PLGS folders back up any databanks that are stored in the installation directory uninstall previous versions of PLGS Backing up the PLGS folders Before uninstalling a previous version PLGS make a backup copy of the following folders from your PLGS installation directory docs contains workfl
146. column on the menu To add or remove multiple columns click Add Remove Columns and then select or clear the check boxes for the relevant columns Click OK To change the order of columns Hither 1 Drag and drop the column headers in the table Or 1 Right click the table 2 Click Select Table Columns 3 Click Edit Order Precision 4 In the Edit Column Order Precision dialog box click the column you want to move and then click the up or down arrow Repeat for other columns 5 Click the X in the top right of the dialog box to close Viewing results in the Results Browser To change the precision with which numbers are displayed 1 Right click the table 2 Click Select Table Columns 3 Click Edit Order Precision 4 In the Edit Column Order Precision dialog box locate the column you wish to modify The number of decimal places currently displayed for that column is displayed alongside the column name 5 Click the up or down arrows beside the number Increasing the number results in more decimal places being displayed decreasing the number results in fewer decimal places being displayed 6 Click the X in the top right of the dialog box to close Selecting proteins and ESTs from the table To select a protein or EST from the table click the relevant row The peptide table shows all peptides that have been matched to the selected protein or EST The MS spectrum display highlights the peaks matched to peptides fro
147. contains a number of columns and a row for each cluster Rows representing internal standards are shown highlighted in yellow EMRT table Expression Analysis Results Frame EMRT Table 62 go 3 44 7 3 0 lg gamma 1 chain C region 100 0 RA 1 40 j 1 9 1294 629 P08670 Vimentin 100 0 1 04 0 04 0 04 0 99 1742 76 P01842 ig lambda chain C regions 100 0 YAASSYLS 1 57 0 45 0 10 1 00 1501 766 P01834 Ig kappa chain C region 100 0 1 o0 1609 893 P02787 Serotransferrin precursor Siderophi 100 0 1149 647 Q15063 OSTEOBLAST SPECIFIC FACTOR 2 100 0 1796 815 P01834 lg kappa chain C region 100 0 SGTASWC 0 86 0 15 0 08 0 00 669 318 P05092 Peptidyl prolyl cis trans isomerase 100 0 HNGTGGK 2 94 1 08 0 33 1 00 1320 634 P01857 Ig gamma 1 chain C region 100 0 STSGGTAA 1023 551 P51884 Lumican precursor LUM Keratan s 100 0 FNALQYLR 644 423 P02768 Serum albumin precursor 100 0 LDELR 0 88 0 1 61 0 62 1109 594 PO1009 Alpha 1 antitypsin precursor Alpha 100 0 LSITGTYDLK 1649 91 P07355 Annexin II Lipocortin II Calpactin 100 0 SALSGHLE 1075 518 P01009 Alpha 1 antitrypsin precursor Alpha 100 0 LSSWVLLMK 977 539 P02787 Serotransferrin precursor Siderophi 100 0 DGAGDVA 1332 759 P35227 DNA binding protein Mel 18 Zine fin 1377 697 1311 725 1073 578 1251 665 1417 672 1762 9 2140 053 1250 619 614 284 1890 942 1127 671 1285 617 1640 941 1576 806 1282 644 1296 655 194
148. contents of the list are displayed in the right hand side of Sample Manager The samples are added to the Sample Manager tree see Annotating and tracking samples with Sample Manager on page 4 1 5 3 5 4 Sample list requirements Rule MassLynx sample lists are not suitable for importing into PLGS There are requirements for any sample list that you will import into PLGS It must bea text file Columns must be either comma separated or tab separated If columns are comma separated the file extension must be csv If columns are tab separated the file extension must be txt Two columns must appear in the sample list Sample Name and Data Path Required columns in sample lists Column name Description Sample Name The name of the sample It can be either an existing sample in the current project or a completely new sample Data Path The path to either a raw data folder or a processed data file xml pkl or txt Additionally PLGS recognizes several other columns which you can optionally include in the sample list Optional recognized sample list columns Column name Raw Data Location Description If the Data Path column refers to raw data paths then this column will be the IP address or name of the computer the raw data is located on If this column is not present in the sample list then it is assumed the raw data is located on the local machine Workflow Template The
149. d digest To run a simulated digest of the current protein or EST right click the Protein Workpad click Digest fragments and then click a digest reagent from the list Result A table is displayed showing the fragments produced by the simulated digest Results browser 6 29 Protein Workpad digest fragments Protein Workpad x Sequence IGYK 479 27 5 8 m KOLK 515 34 82 85 m QVLY 521 28 56 59 m HGRY 531 26 132 135 O CGVIF 537 26 28 32 m MLEW 551 22 1 4 O LNPSK 557 32 23 27 m QENW 575 23 98 101 O CDTCR 596 2 189 193 O DTFHF 665 28 183 187 O APIPLY 672 38 76 81 O KCDTCR 724 3 188 193 m WOENW 761 31 97 101 im ALKDAIF 776 44 176 182 m ETGIPTY 779 37 143 149 O QLVGDDF 792 37 36 42 J APIPLYK 800 48 76 82 m LYSNLSW 817 43 194 200 O DAIFDTF 827 37 179 185 O LAGEIEK 829 45 136 142 O CGVIFDGF 856 38 28 35 m O Retrieving databank entries Use a bookmarked sequence databank search tool to retrieve the databank entry for the current protein or EST To carry out the search right click the Protein Workpad click Bookmark and then choose the Web based sequence retrieval system to use The results of the search are displayed in a browser window To add more sites to the bookmarked list use the Bookmarks tab in the ProteinLynx Browser Preferences dialog box see Bookmarks tab on page 2 11 6 30 Viewing results i
150. d from the search BLAST results When the search is complete the results are returned in a BLAST results panel The BLAST results panel is added to the results desktop which is common to this and other ProteinLynx tools In the example illustrated the results panel displays the hits obtained by submitting a single sequence for BLAST searching 14 26 Query Tools BLAST results panel m BLAST results for workflow BLAST Results Summary Peptide Number Peptide Sequence Number of Hits 1 RESPWRNR 10 2 ERSPWRNR 10 3 RESPNLG EK 10 4 RCLGPNLEK 10 RESPWRNR Accession Description Length E Value External Number Links 029063 YB99_ARCFU Hypothetical protein AF1199 447 3 28384 SWISS PROT TrEMBL NCBI Entrez YADE_SCHPO Hypothetical 40 0 kDa protein 24 1643 SWISS PROT TrEMBL NCBI Entrez MALZ_ECOLI Maltodextrin glucosidase EC 24 1643 SWISS PROT TrEMBL NCBI Entrez P527380 SYG_LUPLU Slutaminyl tRNA synthetase E 24 1643 SWISS PROT TrEMBL NCBI Entrez UBIG_XYLFA 3 demethylubiquinone 9 3 meth 24 1643 SWISS PROT TrEMBL NCBI Entrez P47897 SYQ_HUMAN Slutaminy tRNA synthetase E 24 1643 SWISS PROT TrEMBL NCBI Entrez P20372 PCXB_ACICA Protocatechuate 3 4 dioxygena 24 1643 Navigating within a BLAST results panel The BLAST results panel consists of an upper and a lower section The upper section lists the sequences which have been BLAST searched Click a Peptide Sequence hyperlink in the upper section of the window BLAST
151. d processing acquired data files A 2 MALDI test procedure A 5 Acquiring Q Tof MSMS data A 14 Adding a new databank A 25 A 1 Creating a project and processing acquired data files For further information see Chapter 5 Specifying samples vials and plates with Container Manager Setting samples To set samples 1 oS ue ee a Click Sample Manager Note Sample in this context refers to a batch or bottle of analyte as distinct from a single RAW file or line on a MassLynx sample list Click File gt New Project Type a project name and then click OK In the navigator tree click Original Samples and then right click Click Add New Sample Click No to the question Add new sample to vial Rule For MALDI the Target Plate container type is used instead Annotate the relevant fields with any required sample information To input information click the required field and then type in the text box Tip The text box is active even if no flashing cursor is visible Setting the target plate A 2 To set the target plate 1 2 3 4 Click Container Manager Click Target Plates and then right click Click New Target Plate Type a title for the plate Requirement For MALDI HT this should match the barcode on the plate to be analyzed Quick Start Tutorials 10 11 12 13 14 In the navigator tree expand the Target Plate node and then click the plate you created Drag ac
152. djust the way that the Real Time system processes data click Settings gt Real Time Processing You can set the following parameters Real Time processing setup parameters Parameter Start Processing After Description The real time system will remain idle until the acquisition time has reached this value Example If you only expect peptides to elute after 10 minutes set this value to 10 Check for new peptides every Set this time to determine how often the acquiring data is to be processed Example If this is set to 20 seconds then the raw data will be processed every 20 seconds and if any further peptides are found they will be submitted to the microkernel search engine Remote searching It is possible to process data on the acquisition PC and submit processed spectra to a search engine running on a remote PC This can be particularly important if the acquisition PC on which MassLynx is running is of limited power To set remote searching 1 Click Real Time gt Disable Real Time Processing 15 14 Real Time Databank Searching Click Settings gt Microkernel Search Engine Select Microkernel Remote to enable the Microkernel URL text box Type the URL of the computer on which the microkernel search engine is running and then click OK You should ensure the microkernel is running on the remote PC On the remote PC start the microkernel automatically by starting ProteinLynx browser
153. e applied Intensity Threshold MALDI MS MALDI PSD MX only The number to be used when locating the lockmass peak De isotoped peaks with intensities below this threshold will not be considered as potential lock masses Set the units for this in the Threshold Type attribute Threshold Type MALDI MS MALDI PSD MX only Select how the Intensity Threshold attribute is expressed BPI A percentage of the base peak intensity Counts A specific number for the threshold 8 7 8 8 Mass Accuracy attributes Continued Attribute Perform Lock Spray Calibration Applies to Electrospray Survey Description Enable or disable Lock Spray calibration Enable for data acquired using an external Lock Spray interface Lock Spray Lock Mass Electrospray Survey MSMS Electrospray MS Low Energy High Energy The expected position of the external lockspray peaks Example For a doubly charged species with molecular mass 1569 6696 Da this is 785 8426 Da e The Electrospray Survey value preferably doubly charged will be used to correct survey data and the MSMS value preferably singly charged will be used for fragmentation spectra Rule The same lockspray function is used for survey and MSMS If only one lock spray ion is present the same value can be entered in the survey and MSMS boxes Lock Spray Scans Electrospray Survey MSMS Electrospray
154. e displayed in the Data Directed Analysis Status To acquire data 1 Inthe main MassLynx window click gt to open the Start Sample List Run dialog box 2 Select Acquire Sample Data and Auto Process Samples 3 Click OK A 21 Data Directed Analysis chromatogram displays Chromatogram Sample_010 E Fie Edit Display Process Window Tools Help x Sapori aano a aeRO amp amp a ne gt 8 5 TOF MS ES BPI 222 4 TOF MSMS ES 750 455 BPI 37 525 835 460 785 436 749 690 863 3 TOF MSMS ES 758 412 cS 500 950 29 467 303 476 797 607 352 2 TOF MSMS ES BPI 67 573 368 445 262 505 949 1 TOF MS ES 9 00 10 00 11 00 1200 13 00 A 22 Quick Start Tutorials Data Directed Analysis Status display Data Directed Analysis Status a x Function Description Current State 1 Survey Scan STOPPED 2 MSMS Scan of 544 3668 RUNNING 3 MSMS Scan of 565 3564 RUNNING Stop MSMS At the end of data acquisition Peptide Auto begins processing data information This is displayed in the PeptideAuto Server window see Figure titled PeptideAuto Server display on page A 12 The MassLynx sample list page shows the status of the instrument Instrument status in MassLynx KA MassLynx global server qtof micro SPL File View Run Help 2 OBA SD D BADD Zhou B aueue GF status qtof micro Samples 1 to 1 Sample 1 Processing Spectrum Chromatogram Map Edit S
155. e variable 2 Click the sample variable or are grouped together attribute by which you want to group Custom attributes are included in this list 3 To group by more than one attribute so that samples which have the same values for Condition and Sex are grouped for example use Ctrl or Shift to select multiple attributes 4 Click Apply Manually assign 1 Click Manually assign Samples are grouped sample groups sample groups manually according 2 Click Apply and then fillin to user defined the details in the Manually variables Define Experiment Variables section described below Manually Define Experiment Variables Use this section to define the variables you will use to group the samples Rules This section applies only if Manually assign sample groups is selected in the Select Grouping Method section If manual group assignment is selected at least one variable must be defined for each experiment To create a new variable 1 Click New 2 Inthe Variable box type a name for the variable 3 In the Values box type a value for the variable 10 6 Using Expression Analysis to compare and analyze sample groups 4 Click Add To add values to a variable 1 Click New 2 Inthe Variable box select the variable you wish to add a value for 3 Inthe Values box type a value for the variable 4 Click Add Manually Assign Samples To Groups Use this section to assign
156. e XSL style sheet for a particular search tool is required to define which of the results that it receives from a prior search will be used to formulate its query Default filters for AutoMod analysis AutoMod_filter xsl and De Novo sequencing DeNovo_filter xsl are provided These two filters are sufficient for the majority of workflow templates AutoMod filter The default AutoMod filter discards proteins that have a score less than zero Therefore only proteins with scores above zero undergo a theoretical digest and subsequent modifications substitutions and deletions De Novo filter The De Novo filter enables the default threshold values of different parameters to be altered through the browser without having to modify the XSL document The filter provided enables the ladder score and precursor mass thresholds to be amended The ladder score is based on the number of b and y ions in the peptide The more b and y ions there are the higher the score The more consecutive b and y ions the higher the score y ions also contribute a greater score up 66 than b ions In the following example only the MS MS spectra of precursor masses greater than 1000 Da that have not matched a peptide with a ladder score greater than 70 will be submitted for sequencing De Novo Query Filter parameter Wi ProteinLynx Browser File Edit View Options Tools Rae Poa DAROT amp __ Tools EREA BLAST Searching m Workflo
157. e choice for Fragment Ion databank searches containing peptide sequences as it means that the sequences are not digested Non specific will digest sequences non specifically resulting in longer databank search times This is a suitable choice for all databank search types PMF Fragment Ion search and so on although a non specific digest can be more suited to AutoMod analysis see AutoMod Analysis 14 9 tool on page 14 14 where a small subset of databank entries can be submitted for characterization A non specific digest reagent generates all the possible peptides up to a length of 30 amino acids for each databank entry It is recommended that you do not select a non specific reagent without the use of additional filters due to the large number of theoretical peptides that will be produced Rule If an AutoMod search is part of a search sequence and a Non specific digest reagent is specified all proteins will show 100 missed cleavages irrespective of which digest reagent was used in the preceding databank search step For a PLGS search to add alternative reagents to the existing list use the Digest Reagent Tool see Getting started with the Digest Reagent tool on page 12 7 For Mascot searches see your Mascot server administrator Secondary Digest Reagent This attribute is optional as a default reagent is supplied If two digest reagents are applied to a sample they are applied sequentially Therefore a theoretica
158. e fragmentation spectrum for the peak that is matched to the peptide is displayed The graph is annotated with the fragmentation data for the peptide Peptide fragment annotation indicates the peaks that correspond to fragment ions from the peptide and marks the positions of these ions within the peptide sequence The colors in the graph are Red y series ions Blue b series ions Green All other ions Displaying ion probabilities To display ion probability data for the fragmentation spectrum click the button to the right of the spectrum view To view data for one or more ion series select each relevant check box on the display 6 22 Viewing results in the Results Browser Q 5 w 8 M MSMS spectrum ion probabilities is jb Ja y b H20 Jones My v H20 Jez Chart Y mass error i influence Influence bits For each matched fragment ion you can view either mass error or influence or both Mass error is the difference between the theoretical mass of a fragment ion and the peak mass from the spectrum to which the ion was matched The peptide sequence is shown along the bottom of the graph and each ion is indicated by a colored dot above the relevant position in the sequence The color of the dot indicates to which series the ion belongs The vertical position of the dot indicates the mass error To view the mass error for the selected ion series select the check box labeled mass error To
159. e local machine and are administered by the local search engine Databanks that reside on a remote machine and are administered by a remote search engine can only be viewed not edited To edit a databank 1 Click the databank in the navigator tree The Databank Editor Panel is displayed Databank Editor Panel Databank Admin Tool Q GH Databanks LS Databank Attribute Name Format Fasta Format STANDARD_SPACED Location file CPLGS2 2 demodbs s Make Blastable TRUE Index For PepGrab FALSE Load into Memory TRUE Species for Indexing Management Options FALSE Periodically Download FALSE None 30 FALSE None 04 00 13 11 2 Click the required attributes in the panel and then edit them at the bottom of the panel For details of the attributes see Databank attributes on page 13 4 Rule You cannot edit values for the Name or Location attributes 3 Click the Save button to save the databank 13 12 Organizing databanks with the Databank Admin tool Removing and deleting databanks If a databank resides on the local machine you can Remove the databank but not its associated files A removed databank can be revived restored later Delete the databank including its associated files A deleted databank cannot be revived restored later Removing databanks from the system record Using the Databank Admin Tool you can remove
160. e mass is an MH value independent of the charge state In Mascot generic format the precursor peptide mass is an observed m z value from which M or MH is calculated using the prevailing charge state Include at least one blank line between each MS MS dataset For more details see www matrixscience com PKL Output PKL format is a Waters file format for storing MS MS spectra The PKL format is similar to the DTA file format but supports multiple MS MS datasets in a single file The first line of a PKL dataset contains the observed m z intensity and charge state of the precursor peptide as a triplet of space separated values Subsequent lines contain space separated pairs of fragment ion m z and intensity values Multiple MS MS datasets are delimited by at least one blank line MS Text Output MS Text format is a plain text file listing mass intensity pairs suitable for storing an MS spectrum If this is selected the Top most intense peaks to return check box is enabled 2 22 Setting up ProteinLynx Global SERVER Spectrum Output tab parameters Continued Parameter mzData Output Description The mzData format contains information similar to that in the PKL format but in an open source XML format that is supported by various other scientific software providers See also The Proteomics Standards Initiative s website at http psidev sourceforge net ms To add a fo
161. e masses associated with an excluded item on page 6 34 View Protein Workpad Open the Protein Workpad see Protein Workpad on page 6 27 Hide Workpad Close the Protein Workpad Adding items to the excluded list There are five ways to add items masses proteins and peptides to the Excluded Masses Workpad e To adda mass shown in the peptide tree 1 In the workflow results window click the Show Peptides masses button w 2 From the navigation tree drag the mass you wish to add onto the Exclude Masses Workpad e To add a protein shown in the protein tree 1 In the workflow results window click the Show Proteins button oa 2 From the navigation tree drag the protein you wish to add onto the Exclude Masses Workpad e To adda peptide shown in the protein tree 1 In the workflow results window click the Show Proteins button mi 6 32 Viewing results in the Results Browser 2 Expand the navigator tree to show the peptides you want to exclude 3 From the tree drag the peptide you wish to add onto the Exclude Masses Workpad To add a single mass value to the list 1 Right click the Exclude Masses Workpad 2 Click Add Exclude gt Add Mass 3 Type amass value in the Add Exclude Mass dialog box and then click OK To add a common compound 1 Right click the Exclude Masses Workpad 2 Click Add Exclude gt Add From Library 3 Click the desired item in the drop down list and the
162. e spots again right click and then click Process gt Latest RAW data Results e Progress is indicated on the status bar The interface will be updated as results are returned from the server You can refresh the view periodically by clicking File gt Update A 4 Quick Start Tutorials MALDI test procedure Spot 24 wells of ADH with ACTH lockmass as per the installation specification For further information see Chapter 5 Specifying samples vials and plates with Container Manager Setting the target plate To set the target plate 1 Create a new MassLynx project as described in the MassLynx Help 2 Create an MS Method File MS Method parameters Experiment Setup d plgs2training pro acqudb digestimc exp File Edit Options Toolbars Functions Dslr x alv amp Madi Scan e LMC Total Run _ mm o f Bir 2 2 Male Mass 00 00 to 3500 00 LD O U U l 3 Create anew PLGS project see Importing and viewing PLGS sample lists on page 5 3 Enter the name of the project as PLGS2Training 4 Click Container Manager and create a new target plate as described in Creating a new vial microtitre or target plate on page 5 9 5 Name the target plate Tip If using MALDI HT use the barcode on the plate 6 Anew target plate is displayed Drag over the spots that contain the sample 7 Right click on the selected wells and then click Set Sample see Setting a sample on page 5 11 8 Cli
163. e without deleting the entire databank 1 In the navigator tree expand the node of the databank 2 Click the node of the archive which is to be deleted 3 Click the Delete button 4 on the toolbar 4 Confirm the request when prompted Results e The archive is removed from the Databank Admin Tool record and no longer appears in the navigator tree The underlying zipped archive file is deleted from the file system The archive is not available for future revival Deleting revived archives You can delete revived archives which reside on the local computer To delete a previously revived archive without deleting the entire parent databank 1 In the navigator tree expand the node of the relevant databank 13 14 Organizing databanks with the Databank Admin tool 2 Click the node of the revived archive which is to be deleted E3 Revived archive node dark colored Archive node grayed out 3 Click the Delete button on the toolbar 4 Confirm the request when prompted Results Any files needed for search processing are deleted from the file system In the navigator tree the color of the node changes to gray to indicate an archived databank The corresponding version of the databank is not available to the various search tools The zipped archive file remains in the file system The archive is still available for revival in the future Keeping archived copies of a databank Databanks can chan
164. earch Query search method to identify a set of protein sequences However if you use a Fragment Ion Search only you can also link this method with other search methods Doing so progressively filters the search and analyzes the data more accurately These other search methods are AutoMod Query De Novo Query BLAST Basic Local Alignment Search Tool Query If these are used the results of one search are filtered to form the query of the next This can significantly increase the number of peptides matched to fragmentation spectra data and improves the coverage of the ESTs or proteins in the results You can save the workflow templates for use in other sessions The Workflow Designer interface To open the Workflow Designer click the Workflow Designer icon in the tool tray The Workflow Designer opens with nothing displayed in the main window When you have created a new template the interface contains the following elements 7 2 Defining templates for searching with Workflow Designer Editor panel Displays the attributes for the workflow and search methods Desktop panel Displays workflow templates Workflow Template Displays the search methods to be used for a workflow Workflow node Enables you to attach search methods to create a search strategy Workflow Designer new template Menu bar Toolbar Workflow Designer Workflow Pe Attribute Sample Manager Tit
165. ed expect threshold 14 25 Each search hit returned from BLAST search has an associated E value If searching a randomly generated sequence against a database a certain number of hits would be expected to occur simply by chance The E value of a match is an indication of how many matches of that score would be expected from that databank simply by chance The E value depends on the scoring matrix the size of the databank and the length of the query sequence Low expectation values are a good indication that a hit could be a true hit and has not occurred spuriously The expect threshold is the cutoff value for the expectation values when performing a BLAST search Setting a relatively low expectation threshold gives a stricter criterion for returned hits Setting a high expectation threshold is more lenient with regard to hits returned When searching for short nearly exact matches a high expect threshold is appropriate Gapped If the check box is selected the BLAST search allows for gaps in the alignments in the matching process Low Complexity Filter If this check box is selected the BLAST search masks for repeats in the sequence De Novo analysis of mass spectrometry data typically returns results which are relatively short sequences of amino acids Masking for repeats of such short sequences can result in very little retained data Number of Hits In the text box type the maximum number of hits to be returne
166. ee P Description Reg Score Probability Peptides Coverage Mean Error view Accession Entry E JB001 raw P00722 BGAL_ECOLI Beta galact 116278 1 11 555 TO 2147483648 45 064 2 147 483 648 2 147 483 648 2 1 RTY_0607 A 2 Q9PK92 PKN1_CHLMU Probable s 107430 1 11 062 M 2147483648 11 349 2 147 483 648 2 147 483 648 2 1 BioRad Ge A 3 P28248 DCD_ECOLI Deoxycytidi 21235 1 11 482 3 BioRad Ge A 4 P00370 DHE4 ECOLI NADP spec 48550 6 1 11 555 23 3 BioRad Ge A4 5 Q9Y236 C801_HUMAN Protein C8 56635 7 497 1 7 4881 _ 17 2 8 119 25 345 34 498 Ettan spot Q09910 YAJB_SCHPO Hypothetic 1023 9 341 1 9792 17 4 Eg 7 919 23 961 24 858 JBOOtraw A7 PO0722 BGAL_ECOLI Beta galact 116278 5 411 1 11 555 NET 33 45 064 17 318 61 826 Adjust the size of any column by clicking and dragging the right hand side of the column Change the position of any column by clicking and dragging the column to a new position 6 2 Viewing results in the Results Browser Results browser The workflow results browser displays mass spectrum data alongside results from Databank searches AutoMod analyses and De Novo sequencing The browser can show results from an individual search or merged results from a workflow containing multiple analyses Browser display of results for MS spectrum data m 1Nov HR 4 A 1 ESN 56 hits 171 pro
167. efault the title of the template is the current date and time which is shown in the Editor Panel If desired type a new title in the Title text box The Data Preparation template for each acquisition type instrument has similar attribute sets and attribute panels However the attributes available in the attribute panels depends on the selected acquisition type Click the relevant file icon C in the template to display the attribute panel in the Editor Panel on the left of the screen The details of each attribute are displayed under the attribute panel To save the processing parameters template either Click the Save button on the toolbar or Click File gt Save 8 3 To remove the processing parameters template you are currently editing either Click the Remove button im on the toolbar or Click File gt Remove Note If you are editing an existing template the XML file will not be deleted the displayed template frame and attribute list will just be cleared 8 4 Creating custom processing parameters Attribute sets for data preparation There are seven methods used to acquire data MALDI MS MALDI PSD MX MALDI Q Tof MS MALDI Q Tof MSMS Electrospray DDA Electrospray MS e Electrospray High Low For each acquisition type you can specify the following sets of attributes in the processing parameters templates Mass Accuracy Noise Reduction Deisotoping and Centroiding Pea
168. ein ynx Browser HER Fle Edit insert View Options Tools Ba amp amp B RG w Barr ProjectProteins x ep e Tei Insert paragraph Q Header A Perr m Insert image a L H Horizontal Rule p EO ore Paragraph element Insert horizontal L E Paragraph Gel Manager L Pee j rule amp Horizontal Rule S I introduction Insert field Template Details ts Container Manager j E Paragraph E Paragra Lax paraenn Content buttons HE Paragraph a E Paragraph Expression Analysis ema ie Eine Navigator t a j E Image m Navigator tree Databank Search E oe a L E Field L E Field AutoMod Analysis te fee Page De Novo Sequencing BLAST Searching R Element properties Data Preparation nO0s eae Ea Noobs The four element buttons indicated are available for all types of page Other content buttons become active depending on the type of page selected 3 Add the text You can then use the tabbed pages in the dialog box under the navigator tree to change the position text box dimensions font and text details Tip To center text on a page easily size the text box to the full width of the page and then use the Text tabbed page to set the justification to center 11 20 Creating print templates and printing project data To add i
169. electing for search 14 18 estimated calibration error 14 7 14 21 E value 14 26 Excel files xls 9 4 exclude clusters 10 13 exclude masses 14 11 viewing 6 34 workpad 6 31 Exclude Masses Workpad 6 31 Masses to Exclude window 6 34 executable file for Windows 1 4 Expect Threshold parameter 14 25 Expected Peak Width attribute 8 15 experiment attributes Expression 10 4 experiment setup Expression 16 3 MS 16 3 Export PlugIns 2 23 exporting data 11 2 Expression results 10 12 mass spectra 5 22 projects 3 3 sample list 5 29 A 9 A 19 spectra 5 28 SuperTrack results 5 28 switch lists 10 23 Expression assess data quality 10 25 data 7 2 7 5 16 1 exporting results 10 12 filtering results 10 13 method file 16 3 printing results 10 13 Expression Analysis Design Manager 10 3 Expression Analysis tool 10 2 creating a project 10 2 description 10 2 Expression experiment assess data quality 10 8 attributes 10 4 manually assign samples to groups 10 7 Index 7 manually define experiment variables 10 6 new 10 3 open 10 3 quantitation analysis 10 8 select data 10 7 select grouping method 10 5 starting 10 9 viewing results 10 10 Expression tab 16 5 Expression table opening 10 10 external lock mass 8 6 External Lock Mass attribute 8 6 F FASTA flat file format E 9 FASTA format 14 18 FASTA Format databank attribute 13 5 file format significant clusters 10 24 file formats dta 2 22 mass spectrum 14 5 mstext 2 22 mzData 2
170. engine 2 5 Preferences dialog box Search Engine tab Preferences _ Type Address Connected PLGS 127 0 0 1 true Add Search Engine Type MASCOT v Address 10 62 1 255 80 cgi Description MASCOT search engine on rel Connect vi Detach O ox em ProteinLynx browser can submit searches to PLGS or MASCOT version 2 0 and later search engines running either on the local PC IP address 127 0 0 1 or on remote servers Adding a search engine You can add one search engine of each type PLGS or MASCOT To add a search engine 1 Click Add 2 Click the type of search engine PLGS or MASCOT 2 6 Setting up ProteinLynx Global SERVER T7 Type or paste the IP address of the computer on which the search engine is running into the Address text box To connect to a PLGS server you only have to type the IP address However to connect to a MASCOT server you must type the IP address port number and the path to the CGI Common Gateway Interface directory For example 10 62 1 255 80 cgi Tip Port 80 and 8080 are commonly used for internet applications including Mascot If port 80 or 8080 are not correct please consult your Mascot server administrator The CGI directory contains the program that executes the databank search The default location of this directory is lt IP address gt mascot cgi However it is recommended that you consult your Mascot server administrator to check the location of the di
171. enssssssssrrrresesssseee 1 6 Starting modules manually and troubleshooting problems 0 c 08 1 6 Instaline PLUGS on LINUR gcssssdec si seceacscscsacevsbdccctdcauceacssvacaunvcawssincavecssaxdiansceestes 1 7 Before installing PLGS e essesseeeseereeerseereersseesseeseesssessessessesesssssessssssssssssesssos 1 7 Backing up the PLOS folders noran a 1 7 Backing up daa DARS esar S 1 7 Changing file permissions soriercicseir traii tin nna a E N n 1 8 Uninstalling previous versions of PLGS in Linux sessessssssssssessssssssssssreeee 1 8 es ee PLOSOn LINUX asaran S 1 9 Restoring backed up folders ssssooooooosoossesosessssososessesressroeseosererererrererrreere 1 11 Running PLOS g5 LINU essea AESA 1 11 Starting modules manually and troubleshooting problems 066 1 13 Installing PUGS on UND fja a aaar aaraa aaaea aaea EEE EE ETRS 1 15 Betore installine PLOD 0n UNIK cesna a 1 15 Backing up the PLGO Mirectory isiro renar E ORE aS 1 15 Uninstalling a previous version of PLGS essssssssssssssssssssessererrrsererrrrrrene 1 15 estate PLOG or UNIA ei eae eee 1 16 Cr FLOGS o UNIA cenni a A 1 17 Search engine memory allocation esssssssssssesssesssssssssssssssssssssssessesssesss 1 17 TMPDIR environm nt variable itis cic cstsasiiecinediaeiaewaniaieeiins 1 18 Table of Contents V Search engine temporary directory cicicsissvsevsecavacivassessavdviavinerenesnavevracrnoraas 1 18 Running PLGS on UNIX eraconasri eniin
172. eparated by a space Each subsequent line shows an amino acid from the peptide sequence followed by the mass error for the first selected ion series the influence for the first selected ion series the mass error for the second selected ion series and so on Each entry is separated by a space 6 26 Viewing results in the Results Browser Protein Workpad The Protein Workpad is a separate window that displays details of the currently selected protein or EST To view the Protein Workpad right click either the protein or EST table and then click Protein Workpad Protein Workpad Protein Workpad xi D41 HAEIN Coverage Map MLCWIGYKNG TSPAWQVLYT WOQACDQLOMN YLYR GGOQSL NFL ILPQONSTLY THLQSCSPIH GAVLEQQSLA ES5EKSRCCPS PWLNPSKCGY SGENFAPIPL EISDHQSTLS CGANWALKDA IFDGFQLVGD YKQLENQPHL KHGRYLAQEI IFDTFHFKCD DFNSDQTAEN SQDLIKWQEN EKETGIPTYY TCRLYSNLSU EE _ Key Regions of the protein sequence that match peptides are highlighted in colour according to the key below Matched to a peptide Initially the protein workpad shows a coverage map of the currently selected protein or EST see Coverage map on page 6 28 To change the view right click in Protein Workpad A pop up menu opens Results browser 6 27 Protein Workpad pop up menu Digest Fragments gt Bookmark gt Hide Workpad The menu items are Coverage Map shows the protein sequence and pep
173. eptideauto Port 9878 Blocking Mode results v none Processor spectrum Host 127 0 0 1 Port 9877 You can set the following parameters Parameters tab parameters Parameter MassLynx Directory Description Type the pathname of the directory in which MassLynx is installed on the local PC PeptideAuto Port The port enables the application to interface with other modules Type the port number used by the PeptideAuto module PeptideAuto handles submission of data for processing and workflows for searching from MassLynx Recommended Use the default port number 2 19 Parameters tab parameters Continued Parameter PeptideAuto Blocking Mode Description The blocking mode parameter describes the data acquisition behavior of MassLynx The following blocking modes are available none MassLynx will continue to acquire data while previously acquired data is being processed or used for searches spectrum MassLynx data acquisition will be blocked until any previous data has been processed although data can still be acquired while previous data is being used for searches e results MassLynx data acquisition will be blocked until any previous data has been processed and until any searches using the previously acquired data are complete Recommendation The preferred option depends upon the hardware configuration For exam
174. er V Container Manager 8 Expression Analysis Databank Search AutoMod Anal 2 De lovo Sequencing BLAST Searching Data Preparation 5 ATF Print Tool Print Tool D SampleProjectTemplate D HeaderFooter C Header L E Paragraph C Footer im D introduction C Template Details LH image D Results Project content C Locations content C Hits content Proteins content L C Peptides conte L Summary Typeface Arial Element Properties ggnanOdcC OTMEAnAE Field Element with Drop Down List 4 Use the tabs to change the font and dimensions of the field Buttons for adding content to pages You can use the buttons on the right of the browser to add content to a page The first four buttons for adding paragraphs images horizontal rules and fields are available for all pages Whether the other buttons are available depends on the type of page selected which controls the type of content that can be added The buttons enable you to drag a rectangle to the required size anywhere in a page 11 23 The details of the buttons are shown in the following table Print Tool buttons for adding content to pages Button E Function Inserts text box for a text paragraph Available for all pages mT A Inserts an ima
175. er of Processors lt number gt Where lt number gt is the number of processors you want Expression data processing to utilize 4 Save the file Databank searching Recommendation Make a copy of the file before editing as making changes other than those explicate outlined below could prevent PLGS from operating properly To set the number of processors for databank searching 1 Navigate to the config micro directory underneath the PLGS installation directory 2 Open the mkconfig file 1 25 The file contains the following lines 0 0 1 8 100000 8192 config micro mod_ list txt config micro BLOSUM62 txt Number of 1 Processors 3 On the seventh line of the file type the number of processors you want databank searching to utilize 4 Save the file 1 26 Installing ProteinLynx Global SERVER Setting up ProteinLynx Global SERVER You can set up the ProteinLynx Global SERVER browser for the way you want to work this includes Adding and removing tools from the Tool tray Identifying search engines processors and instruments that are to be used to process data Specifying Uniform Resource Locators URLs for Web sites that can be referenced within the application Setting the colors for the display of the microtitre and target plates Setting the style and display for printing results Specifying the location of modules used in automated processes and
176. es with Container Manager Browser displaying processed data Ya Protein_ynx Browser File Edit View Windows Options Tools B 18g OBE Container Manager H B96 vials Processed j F Microtitre Plates mass a n Target Plates spectrum RES Default QTOF MSMS CE E EE E E ES a Oa a 3 2 2 ae 0 207 node AA CAMassLynxProteinLyr T I pnt i Default ATOF MSM Processing Parameters amp Senn template Processing Pafameters Template 8 forkflow Templates z Workfl OW Precursor mass 682 419 charge 3 template 2628 2205 max HHE ME Viewing the mass spectrum Data from a processed mass spectrum node can be viewed in the Processed Data Viewer To view the processed spectrum 1 Click a processed Mass Spectrum node and then right click 2 Click View Spectrum Result The Processed Data Viewer displays the processed spectrum with a list of corresponding monoisotopic masses 5 19 Re searching processed data To add more workflow templates to the processed mass spectrum node 1 In the navigator tree click the processed mass spectrum node that you wish to add a workflow template to and then right click Click Attach Workflow Template Click a workflow template in the drop down list or click Choose new workflow template from file If you have selected to choose a new template browse to the template in the Select
177. escievssesseseisussesusseaiesavesiocesesassussessssedouerecgusedessesveceeses 11 3 POPE PEDT IG na ess does ees eeiasanie ies Glaeser anes eas 11 3 Work on DOLS eaaa venta teenie Neneien 11 6 Opening and deleting print templates ssssssssssssosssosssossoossoossossossssossosseso 11 12 Creating print templates ssessesesseseceeccceccceccecccecccecceeeseeeseeseeesseeseesssesseesssoss 11 13 Adding content to the results nodes sssccccscisrsiiriiassninisiississsis 11 15 Filtering sorting and limiting in results nodes ccccccceeeesstteeeeeeeeeeees 11 16 Alert ecards ae renee Emm re Pear Ne Pen seem ODT rato ot eset Fen eee ere ae tr te er ealee ener 11 16 Pe VES a A 11 17 Baragaon ees ecard E PEE OE ETA 11 17 Customizing print templates ssssessssessessseoseossecoseooeosoecoecoceecoeeoeeeceeeoseesseeoe 11 19 Buttons tor adding content t pages eeroeusiv isins Aana 11 23 12 Managing modifier and digest reagents ssessssseecsseeceeooseoossssosssse 12 1 Getting Started with the Modifier tool sssssessesssoossoosecoecoceocsecoeeooseoseeoee 12 2 Viewing existing modifier reagents ccccccccccecceccceecscccscecsccseceeseeseceeseeees 12 3 Adding and editing custom modifier reagents ccccccccceccssecseccesceseeees 12 4 Deleting custom modifier reagents eseserreereererrereeresrerssresresssesssessssssss 12 6 Getting started with the Digest Reagent tool cccccccceccseccececseeseceess 12 7 Viewing exi
178. est reagents Custom digest reagents You can add edit save and delete custom digest reagents Adding or editing custom digest reagents To add or edit a custom digest reagent 1 To adda reagent click the New button ay or click File gt New lt To edit an existing custom reagent click the reagent in the list Rule Existing custom digest reagents are shown in black text in the list For both actions a panel and text box are generated which enable defining or editing of the values for each attribute Adding a new digest reagent Edit Digest Reagent Attribute Value Name Specifier Name Enter the name of the digest reagent Name Rule Only user defined digest reagents can be edited you cannot edit the supplied reagents 2 Click a row in the panel to update the value of the attribute You can amend the values for the following attributes New digest reagent attributes Attribute Description Name Type a unique descriptive name 12 9 New digest reagent attributes Continued Attribute Specifier Description Edit this attribute to specify the cleavage points and exclusions of this reagent The syntax of the specifier is as follows forward slash indicates a cleavage point back slash indicates an exclusion for that cleavage for the C terminus hyphen then back slash indicates an exclusion for that cleavage for the N terminus S
179. eteedecovevectovecteeccosececsterds 14 23 BLAST search parameters s c i iidsosccsdecsaeancnsrocsstosvaietocantagsnteninetedsataonaicdevnnses 14 24 Peptide geqn nte senromansk a aA 14 25 GI a E 14 25 Hapert Thre ol arni E 14 25 E ETE o E EE E T T A E I E E E A AE 14 26 Low Complesity keene permenant eaaeee 14 26 UR ot HE eaaa R neta aeons 14 26 Table of Contents BLAST POI e AOE ETENE 14 26 Navigating within a BLAST results panel esssssssesssessssssesssrssssssrrreeee 14 27 15 Real Time Databank Searching eeesssseseeoossssossssseesseeeeeeeoeeeccesssessse 15 1 Using real time databank searching sessessssessesssssssesssssssssssossesssosssessossso 15 2 Launching the Real Time Databank Searching application 05 15 2 Processing parameters sssccscsecisseorsnevavsnversceresunvasvnvsvensevedursesdavencrveraievenneveds 15 4 Soare hine PIA OE a a 15 5 RESIDES OTIR d erent ee oer PE eno ened ner me PEs ne eet nPDOP poe Pee er ETS 15 7 Setting up a real time databank searching acquisition cccccceeeesseeees 15 8 Derne Tip rout DDA ide ics si esd endeared 15 10 De isotope peak detection wsicwiciiniawissinnmicinainainnGasnanwadens 15 11 Tolerance window sii cinet icine ease eas ees 15 12 Estacion WO nicer eee 15 12 Esc hide WR science es 15 13 Other DDA experiment Settings ccccccccccccccccccceccceccseececcesecceecesceseeeeeeens 15 138 A AVANCE options ssis seinni rE EEEa EEE EEEE sensed E SNRA 15 14 D
180. ets from within the template navigator tree To add content from the template navigator tree 1 Inthe template navigator tree right click the Results node or one of the content nodes 2 Click Insert gt Content Page 3 Select a results table and then click OK Result The content page is added to the navigator tree below the selected node 11 15 Filtering sorting and limiting in results nodes You can filter sort and limit the results in content pages In the navigator tree click a content page Properties for the content page are displayed in the lower part of the pane Filtering results To add filters click the Filtering tab and then click Add Properties dialog box adding a filter GRIEG Sorting Limiting Add VA Edit Filter AA Sequence Edit Remove Up Down Range from D Boundary Greater than Enter regular expression Select Boolean Match True v The drop down menu contains all the fields available to filter the results Different options are available in the dialog box depending on the field selected Numeric Range and Boundary options are enabled Text Enter regular expression option is enabled Curated Select Boolean Match is enabled The Combine and Add options enable you to either combine this filter with other filters or use this filter in additi
181. ew project To create a project 1 Inthe tool tray click the icon for one of the tools that requires a project Sample Manager Gel Manager Container Manager or Expression Analysis 2 Click the Create new project button Ce on the toolbar 3 Type a name for the project 4 Click OK Result The Container Manager window looks similar to the following illustration Container Manager with new project zi ProteinLynx Browser File Edit View Windows Options Tools Container Manager My project 868 vials Microtitre Plates Sample Manager an Navigator tree Ea Processing Parameters Templates Workflow Templates Gel Manager Container Manager Expression Analysis Data Preparation EDY Workflow Designer nO006 Container Manager 3 2 Creating importing and managing projects Importing and exporting projects To import a project 1 4 In the tool tray click the icon for one of the tools that requires a project Sample Manager Gel Manager Container Manager or Expression Analysis Click File gt Import Project Click the Files of Type drop down list and then click the type of project file you want to import PDQuest XML Sample list XML file generated from PDQuest software Importing this file type imports any gel container and sample tracking information specified in the XML Progenesis XML Experiment XML file generated from Progenesis Discovery software
182. ext to open a template selection dialog box Recommendation New users should use default templates 4 Click a suitable template and then click Next Project print wizard Choose a Print Procedure Ji Print Project Edit Limits Proteins table In this screen you can print immediately preview the report see Figure titled Previewing a project report on page 11 5 or export the data toa csv or html file type Recommendation It is reeommended that you preview the report The Edit Limits dialog box enables you to override the limiting options for the results that are set in the template see Limiting results on page 11 17 However the settings in this dialog box are not saved in the template 5 After selecting an option click Finish 11 4 Creating print templates and printing project data Previewing a project report Ji Page Preview ProjectProteins 3 a E 0 enas Canteen Coreen 1 ro dee Prasan Decree Coverage Foptiinn Cavittonce Scare r m FOr Conpannerrc J procuraor Core aire tae Te aes Waters oaeroriens aaa lasna anamata aerae aTa tT ma Tacan riabercCoreter tt gt gt im Row Ooi Caanar Corenar 2 ae fom t n Prosen Doscrpein il coma Cantiano A Paport Datena Canana Corana 4 A AOIN PEAT DOO 100 Canana Corema 5 one ona acron The report siaaa tha top 2 protana found in each container pasti i tha sacle projact This tampia iaa NOT show any prefers ud
183. f its children and stores them for use in the paste operation Edit gt Copy Copies the selected node and all its children Edit gt Paste Attaches the previously copied cut node hierarchy to the position selected iT Edit gt Delete Deletes the currently selected workflow node and all its children He Options gt General ProteinLynx preferences Els Preferences 7 4 Defining templates for searching with Workflow Designer Creating a workflow template To create a new workflow template 1 2 Click G on the toolbar A panel is displayed which enables you to select a search type for the template Workflow Designer selecting a type of search Workflow Designer Create A New Workflow Template Select Search Type PMF PNF Fragment lon Search Fragment lon Search Electrospray MS Electrospray High Low O Q Select a search type and then click Tips Fragment Ion Searches can be performed from any instrument that can generate fragmentation spectra Therefore Fragment Ion Searches can be performed on Electrospray Q Tof Maldi PSD and Maldi Q Tof data The Electrospray MS option enables searching of low energy MS data only effectively a peptide mass fingerprint The Electrospray High Low option enables searching of both the low and high energy MS fragment data 7 5 Result A new workflow template containing a new wor
184. f screen areas 8 3 processing parameters templates 8 5 removing processing parameters templates 8 4 saving processing parameters templates 8 3 select data type 8 2 data quality viewer 10 25 data type MS 7 2 MSMS 7 2 PSD 7 2 Databank 14 1 databank archives reviving 13 15 attributes 13 4 Databank Admin tool 13 2 13 2 13 17 description 13 2 databank attribute Download Compression Type 13 8 Download Renew Period 13 8 Download URL Address 13 8 FASTA Format 13 5 Format 13 4 Index For PepGrab 13 6 Keep Archives 13 10 Load into Memory 13 6 Location 13 6 Make Blastable 13 6 Management Options 13 7 Name 13 4 Periodically Download 13 7 Periodically Update 13 9 Processing End Time 13 10 Processing Start Time 13 10 Species for Indexing 13 7 Type 13 4 Update Compression Type 13 9 Update Renew Period 13 10 Update URL Address 13 9 Databank Search 14 3 14 13 automated task 7 8 parameters 14 5 tool 14 3 14 13 Databank Search parameters 14 5 Data File 14 5 Databanks 14 6 Database 14 6 Enzyme 14 9 Estimated Calibration Error 14 7 Exclude Masses 14 11 Fixed Modifications 14 10 Fragment Tolerance 14 7 Instrument Type 14 13 Mass Spectrum 14 5 Mass Values 14 12 Maximum Hits to Return 14 9 Minimum Peptides to Match 14 9 Missed Cleavages 14 10 Molecular Weight Range 14 8 Monoisotopic or Average 14 12 MSMS Tolerance 14 7 Peptide Charge 14 12 Peptide Tolerance 14 6 pI Range 14 8 PLGS 14 4 Primary Digest Reagent 14 9 Prote
185. f space allocated to a particular mount point Enable LARGE_FILE support for the mount point This can be done using the system administration tool Remove limits on memory allocation for a user account This can also be done using the system administration tool If you are unsure how to perform these tasks check with your UNIX administrator 1 22 Installing ProteinLynx Global SERVER Restoring old databanks When performing a new installation any databanks added to previous versions are not available from the new PLGS version The databanks must be restored using the Databank Admin tool This tool allows you to specify the format of the databank usually FASTA and the sub format of the databank such as NCBI_EXPASY_GENERAL Caution If an incorrect databank format is specified the databank will not be added correctly which can subsequently cause problems with PLGS To determine the type of databank view the first line of the databank in a terminal window by using more lt databank name gt For information on the various formats available see FASTA flat file format on page E 9 1 23 Setting the number of processors If the computer on which you are installing ProteinLynx Global SERVER has more than one processor you can take advantage of the additional power with PLGS Tip If your computer only has one processor or if you wish PLGS to only use one processor you do not need to make any changes The
186. fe X Attribute Day Value Tuesday Month March Sample Manager Viewing PLGS sample lists Once a sample list has been imported you can view the list and modify certain aspects of it You can also use the list to view the spectra and workflow results associated with a sample 5 5 The sample list table provides an alternative to the navigator tree for viewing editing and processing the data in a sample list To open the table for a sample list click the sample list in the navigator tree right click and then click View Sample List Table Sample List table ms SampleList txt jauto01 Default MALDI MS v v Jauto02 Default MALDI MS IMyfirstworkfowtemplate W auto03 Default MALDI MS w Data either raw or processed that is associated with a sample in the sample list is represented as a single row in the table There are several columns in a sample list table Sample list table columns Column name Description Sample The name of the sample Raw Data The name of the raw data Cells in this column have tool tips that display the full path to the raw data where appropriate Processing Parameters The name of the processing parameters template Template attached to the raw data If the data represented by a row is processed this column is empty Workflow Template The name of the workflow template most recently attached to the data If there is no workflow template a
187. flow templates e Chapter 8 Creating custom processing parameters for details of processing parameter templates 9 8 Viewing and processing gel data with Gel Manager Viewing gel data Viewing a gel image A gel image can be viewed by clicking the node of the gel in the navigator tree The image can be manipulated in the following ways If gel spots have been imported for the gel the spots will be circled to mark their locations on the gel image To remove these circles right click the image and then clear the Circle Gel Spots check box Right click the image and then select the Show Axis Labels check box Labeled axes for the image are displayed Zoom in to a region of the gel image Select a region of the gel image by dragging a rectangle on the image Zoom in to the selected region by double clicking inside the rectangle Repeat the procedure to zoom further into the image To zoom out double click the image without selecting a rectangle first Select a gel spot by double clicking the gel spot on the image or by selecting the gel spot icon in the navigator tree If workflow results have been obtained for the sample from the gel spot the name of the top scoring protein or EST from the search results is displayed when the mouse is moved over the gel spot Viewing a summary of results for a gel Click the Gel icon in the navigator tree to view a gel summary The summary tabulates the top scoring protein or E
188. for Workflow 7 3 03 for 4 1 on Sample plate 1 i Hemoglobin alpha Gel Manager rocessing Parameters Te orkflow Templates Container Manager Expression Analysis max N o 1763853 2 Counts 22 2 BE Q Databank Search Precursor mass 1226 6039 charge 1 H Fk 4 o cc mx kN tetee LIDEK E F yna f f 129 102911 fe 051 4441 L ye l 2 EE 1 1 1 1 1 AutoMod Analysis 10071 0682 Counts o HEREROENE ax Q o amp A 24 Quick Start Tutorials Adding a new databank For further information see Getting started with the Databank Admin tool on page 13 2 To add a new databank Click Databank Admin Tool Click Databanks and then right click Click New Databank 1 2 3 4 5 6 Type a name to use for the databank Set the following fields Type to Protein FASTA Format to STANDARD_SPACED for Swiss Prot or NCBI_EXPASY_STANDARD for the non redundant database nrDB See also Details of the correct format for each database are given in Appendix E Databanks Formats Location click File and browse to the location of the uncompressed FASTA file on disk local or mapped Make Blastable to FALSE this option creates a BLAST Basic Local Alignment Search Tool compatible copy of the database on disk and is required only when sequence data is available Load into Memor
189. for the sample 22 01 03 14 13 33 INFORMATION Removing unwanted temp files 22 01 03 14 13 33 STATUS Setting qitem member variables 22 01 03 14 13 33 INFORMATION Adding item to processor queue 4 The data can be viewed periodically in the main PLGS window as it is acquired To view this data in PLGS click either File gt Update or on the toolbar All the latest results are displayed in the browser 5 32 Specifying samples vials and plates with Container Manager Troubleshooting failed client server workflows If workflow queries sent from a client machine are failing for an example failed workflow icon see Workflow and spectrum icons in the navigator tree on page 5 18 check the following Check that the client is connected to the correct PLGS server If you have recently installed the client software you need to re add the server using the ProteinLynx Browser Preferences dialog box on the client For details see Changing preferences on page 2 5 To add a new server to the list type the IP address in the text field at the top of the dialog box and then click Apply Any errors displayed are usually because the PLGS server components search engine microkernel are not running on the specified computer e Check that the workflows are referencing a databank that exists on the server you are connected to Check this by opening the workflow template in the Workflow Designer see Opening workf
190. g box opens This dialog box has three tabbed pages any of which can be used to select the color Swatches Enables you to select from a panel of predefined colors HSB Enables you to select a color using the Hue Saturation Brightness HSB color model RGB Enables you to select a color using the Red Green Blue RGB color model Select a Colour dialog box Swatches tab amp Select a Colour Colors selected in this session oOo m E Sample Text Sample Text Original color Sample Text Sample Text Color currently select E m m Sample Text Sample Text y ected Cancel Reset The Recent section shows the colors that you have selected in this session 2 14 Setting up ProteinLynx Global SERVER Select a Colour dialog box HSB tab amp Select a Colour oO E Sample Text Sample Text Original color Color currently selected E E m Sample Tet Sample Text y Lo Cancel Reset Select a Colour dialog box RGB tab amp Select a Colour Graan Lay ie a oe 0 85 170 255 BI e z o A E ti Siac 2 tai 0 85 ao N a Preview oO m W Sample Text Sample Text Original color Color currently selected 2 E m Sample Text Sample Text y ok cancer Reset 2 15 For each page The Preview pane shows how the color selected will look in different situations The top half of the block to the right shows the origi
191. ge box for a user defined image Available for all pages m Inserts a horizontal rule Available for all pages E Inserts a field box for a selectable standard predefined field Available for all pages Inserts a box to display a table for live data Available only for table nodes E Inserts a box to display an MSMS spectrum showing fragmentation data Available only for a Peptides content page Fa Inserts a box to display an MS spectrum showing precursor data Available only for a Proteins content page Gi Inserts a box to display a gel image showing protein separation Available only for a Project content page 4 Inserts a box to display a coverage map showing matched peptide locations Available only for a Proteins content page E Inserts a box to display an influence display showing influences Available only for a Peptides content page ti Inserts a box to display delta masses Available only for a Peptides content page a Inserts a box to display fragment ion data Available only for a Peptides content page 11 24 Creating print templates and printing project data Print Tool buttons for adding content to pages Continued Button Function El Inserts a box to display workflow template parameters Available only for a Workflow content page 11 25 11 26 Creating print templates
192. ge over time as new sequences are added or the databank is periodically downloaded or updated Therefore archived copies of databanks are useful especially if you want to repeat previous experiments using the original databank To keep archives of databanks set the Keep Archives attribute to True when creating or editing a databank see Adding databanks on page 13 3 and Editing databanks on page 13 11 This creates a zipped compressed file of the databank However you must also consider that large databanks create large zipped archive files Therefore consider whether your system has sufficient resources available to store archives Reviving an archive If archives exist for a databank that resides on the local computer these archives can be revived restored for use by the various search tools For example you might want to revive an archive to verify results that were obtained from an older version of the databank 13 15 To revive an archive 1 In the navigator tree expand the node of the relevant databank The available archives appear as gray colored icons 2 Click the archive to be restored Click the Revive button on the toolbar 4 Confirm the request when prompted The color of the node changes z which indicates that the archive has been restored The corresponding version of the Databank is available for searching by the Databank Search tool and if appropriate the BLAST Searching tool The databank ver
193. gine and enabled real time data processing 15 16 Real Time Databank Searching 16 Using MS for qualitative proteomics If a Q Tof Premier instrument is used MS data can be acquired This data can then be used in a protein identification experiment See also M9 data can be analyzed in Expression Analyses configured in PLGS If the optional Waters Protein Expression System is being used analyses can also be configured for MS data acquired from samples without isotope labels See the Waters Protein Expression System Operator s Guide for more details Contents Topic Page What is MS 16 2 Creating an MS method file 16 3 Running an M9 experiment 16 7 16 1 What is MSF If a Q Tof Premier instrument is being used MS data can be acquired When acquiring MS data two MS functions are used in an alternating fashion MS one function is acquired in Tof MS mode at a low collision energy typically 4 eV during which no fragmentation occurs to the precursor ions MS a second function is acquired also in Tof MS mode during which the collision energy is linearly ramped between two user defined energies typically 15 eV to 40 eV This induces fragmentation of any species present in the gas cell at that time Therefore during the time course of the experiment the Q Tof Premier acquires data at low energy before stepping to an elevated collision energy where it performs a collision energy ramp
194. group the group s name is displayed in the Unique column To curate organize your data Rule In the EMRT table curation is possible only on clusters with identification information The following steps apply to curation in the EMRT table In the Protein table only step 3 applies 1 Click the cluster of interest 2 Click the Curate Data button Al 10 11 Result The individual peptide identifications for the selected cluster are displayed in the upper half of Data Curation window The lower half displays the high energy fragmentation data associated with the selected cluster 3 To mark a protein or peptide click the icon to cycle through OK unsure and not OK states 4 When you are satisfied with your settings click x to close the window You can choose to show all clusters those clusters marked as OK or unsure or only those clusters marked as OK To control which clusters are displayed click to cycle through the display modes To view the workflow for a cluster 1 In the results table click the cluster 2 Click the Show Workflows button E Result The workflow is displayed in the Results browser see Viewing results in the Results Browser on page 6 1 for more information To view the replicates for a cluster or protein 1 Click the line in the EMRT or Protein table representing the cluster or protein you wish to view the replicates for eee 2 Click the Open Replicate Viewer button Hi Resu
195. gt willc Beta globin gene from a thalassemia pa D P02647 APA1_HUMAN d nlll Apolipoprotein Al precursor Apo Al gt ville Ig kappa chain C region eens EEE T TE 2489 HE AE 62 3245 P sille Alcohol dehydrogenase I EC1111 YA 1149 6178 2 1148 6100 1148 6078 i O 14 1060 a in C renion 1017 5296 2 1016 5218 1016 5291 0 0073 7 36 9477 97K SU 933 5066 2 932 4988 9326114 0 13 5 3 75 1 3301 IK LETVA a 1763853 2 Counts a 6 gt e E El a B max si Precursor mass e 6039 charge 1 FK a LGEENFK bax Fe Ste ec ee ti Li sias n T 10071 0682 Counts E e EA ax EE EIERODIEIE Results tree toolbar The toolbar below the workflow results tree includes controls for switching between protein and peptide views and also for filtering results to only show those marked in certain ways Results browser results tree toolbar Button Description Switch to protein view 6 4 Viewing results in the Results Browser Results browser results tree toolbar Continued Button Description E Switch to peptide masses view 7 Filter the results to show only those marked with the indicated symbol Clear all protein and peptide OK assignments setting all proteins and peptides to not OK amp o Reset all protein and peptide OK v
196. gtacgtgggttttata tatcatacttc ttgtaatgaactaaaaacgt E 16 Databanks Formats ttcgttcgttcgttatttct tataagcaatat ttcaacttaacaacacaaaa Index Symbols csv 11 2 11 4 11 6 11 9 11 10 dta 2 22 gz 13 8 html 11 2 11 4 11 6 11 9 11 10 jar 2 26 mstext 2 22 mzdata 2 23 olb 5 30 pkl 2 22 14 5 txt 9 4 xls 9 4 xml 7 10 8 4 14 5 xsl 7 11 Z 13 8 z 13 8 zip 13 8 A acquiring data 5 31 A 11 A 21 Electrospray DDA 8 5 Electrospray High Low 8 5 Electrospray MS 8 5 MALDI MS 8 5 MALDI PSD MX 8 5 MALDI Q Tof MS 8 5 MALDI Q Tof MSMS 8 5 Acquisition tab 16 4 ACTH A 5 Add Bookmark dialog box 2 11 add remove columns peptide or protein table 6 14 Add Remove Tools dialog box 2 4 adding databanks 13 3 digest reagents 12 9 export plugins 2 24 gel spots 9 3 gels 9 3 inlet file 16 7 method file 16 7 modifier reagents 12 4 new databank A 25 processing parameters 5 21 sample 4 2 search engines 2 6 workflow templates 5 7 5 20 ADH A 5 AIX installation 1 15 starting PLGS 1 19 algorithm BLOSUM 14 25 PAM 14 25 annotating samples 5 11 Applies to attribute 12 5 archived databanks restoring 13 15 archives 13 10 databanks 13 15 deleting files 13 14 deleting revived archives 13 14 assess data quality Expression experiment 10 8 assess data quality viewer 10 25 associated masses 6 34 6 35 attaching data processing parameters A 8 A 18
197. h a OmuturOnater 6 Aar ia en ities iow magava SCORE vaim Proana are crowed y dacreanng aoma at Cawan Corer 7 ow g Erun ainean prec Ta 31d than aacanety fy dacraanng coverage Suitable for any project targa CaveherCoreter row o Serves atann precurscr rue tr cr amal Adee stows the catana potion for tha pratan matchas 2 Coen Canana orana O Soares 3 Author roma Carano fom Rowe c qae ___ fem teen Omage Pontes jamin Soom 2 un afan precura 7 a Date of parate Dac 21 X4 Ace Prassi Dosentin fi nee Sore moraa Sanas atasan procurar i tooo e Ter Project PON Capanne Jprecursce Core Of m Privet Tamplate PraptPratans POTOA Apne 2 macrogeenan pecura S0475 Cares Pom E Oae 4 2 Ace Povaiion Cantitence Scare Consaier Fum2 Aur es Dru aan precursor Td tat Ace Prose Dencrpsion Coverage Popisa Caritence Scare Foza Serves atann pre 1000 rama FONDAS COCA pecura Conk 512 110 1000 107 x 01023 Apra 2 caacrogeewanprecurscr SADOS 6d rono 7_T7 Carana Fle Canana Fem fom 1 we 3 POITO SAONA MENNO POOSAEA ji 1000 Aee Presen Description Coverage Fopiitoa seare 602768 Sera aban precurscr rono r spr PONS Canaan J precurscy Core T TOSS 1 AAS F01021 Apna 2 reacrogsensn precuracr aa roaa la 423 cam se Arasan Descripsi Caviimco Sean emd S Dan aaran prec fai fet Ace Prana Deacrpinn Caverage Peptitinn Cantitonce Scare Serva aE BH 1000 raat Arnas Conpmaenncaprecucnor Cone OTA
198. h the processor is running In the Description text box type a description of the processor Example Remote processor on UNIX box 2 To connect immediately select Connect If you want the processor to keep running when the ProteinLynx browser is closed select Detach Click OK Modifying a processor You can modify the IP address description and the connection details of a processor To modify a processor 1 3 4 Double click the processor in the list Alternative Click the processor and then click Modify The Modify Processor dialog box opens which has the same fields as the Add Processor dialog box Modify the details as required Click OK Removing a processor To remove a processor click the processor and then click Remove 2 9 Instrument tab Use the Instrument tab to change the current type of instrument This specifies the instrument from which raw data is acquired and can affect various default values for example the default processing parameters used for spectrum data will depend on the instrument type Preferences dialog box Instrument tab Preferences Maldi QTOF MS Maldi QTOF MSMS QTOF MSMS 2 10 Setting up ProteinLynx Global SERVER Bookmarks tab Use the Bookmarks tab to specify URLs for access elsewhere in the system Preferences dialog box Bookmarks tab Preferences Name URL Static BLAST Link Waters http Awww true false Static
199. has been designed for PLGS since ProteinLynx 2 0 The plugin interface provides third parties with a means to import or export data into or out of PLGS The plugin architecture provides a simple interface to external data sources A plugin makes a call back to its associated PlugInHandler in a set order after its run method has been invoked lt Immediately after the plugin has started the handleStart will be called This method provides the required streams to the handler input output and error streams If input to the plugin is to be provided before being acted upon it should be written and the stream closed If a large amount of output is expected it is probably most efficient to perform blocking reads from the output stream until the stream is closed Once the handleStart method has been called it can be followed by calls to handleOutput or handleError handleOutput will be called when bytes are available form the output stream If more output is expected this method should return true handleError will be called when bytes are available form the error stream If more output is expected this method should return true Finally either of handleException or handleEnd will be called but not both handleException if an exception arises which cannot be dealt with using a status code handleException is invoked in place of handleEnd handleEnd if the plugin reaches the end of its task th
200. he first six files selected are attached to the wells If you select six files and there are nine wells files are attached only to the first six wells If a well or spot already contains raw spectrum data a dialog box opens to give you the option to replace the existing raw data However if the raw data has been sent for processing it cannot be replaced a warning message is displayed 5 16 Specifying samples vials and plates with Container Manager Processing raw data 1 To process the data from the navigator tree click the Raw Data Spectrum Node and then right click 2 Click Attach Workflow Template and then click OK to choose a new workflow template from file Tip You might not need to do this if a workflow template was specified in an imported sample list 3 Browse to a workflow template and then click Open The template is displayed in the navigator tree Rule Do not attach a PMF workflow template to Electrospray High Low data See also For more information on workflow templates and how to produce them see Chapter 7 Defining templates for searching with Workflow Designer 4 Click the Raw Data Spectrum Node again and right click 5 Click Process As the data is processed the icons change for the workflow and spectrum see Workflow and spectrum icons in the navigator tree on page 5 18 Also the color of each sample well updates according to the search results see Customizing the plate view on p
201. her value than the one specified Validate Results All MSMS results can be validated A validated peptide will contain a series of three or more consecutive y ions If validation is selected the top scoring peptide for each MSMS spectrum is returned This could increase the requirement for manual validation of the results returned 14 17 Selecting protein sequences for the search Requirement When running a one off AutoMod analysis either protein sequences EST sequences or both must be specified If an AutoMod query is created as part of a workflow protein sequences and EST sequences can be omitted since the proteins and ESTs identified by any preceding databank search are used as the input for the AutoMod analysis Protein sequences can be typed copied and pasted or dragged and dropped into the text area The sequences must be in fastA format Tip fastA format sequences can be added by dragging and dropping proteins from the navigator tree or protein table in a ProteinLynx search results frame Selecting EST sequences for the search Requirement When running a one off AutoMod analysis either protein sequences EST sequences or both must be specified If an AutoMod query is created as part of a workflow protein sequences and EST sequences can be omitted since the proteins and ESTs identified by any preceding databank search will be used as the input for the AutoMod analysis EST sequences can be typed copied and p
202. hide the mass error data clear the check box Rule At least one of the graphs must be displayed at all times if the influence check box was already cleared it will be reselected automatically Influence indicates whether the prediction of the selected ion is having a positive or negative effect on the peptide score the more positive the number the more influential the prediction The peptide sequence is shown along the bottom of the graph and each ion is indicated by a colored bar above the relevant position in the sequence The color of the bar indicates to which series the ion belongs The height of the bar indicates the influence To view the influence for the selected ion series select the influence check box To hide the influence data clear the check box Rule At least one of the graphs must be displayed at all times if the mass error check box was already cleared it will be selected automatically Results browser 6 23 3217 07 max lu To return to the MSMS spectrum view click the button to the right of the spectrum view Spectrum Viewer options Several Spectrum Viewer functions are the same regardless of whether MS or MSMS data is being displayed Viewing a selected X axis range Scrolling along the X axis Displaying a zoomed section of the graph in a separate window Viewing a selected x axis range Rule This function is not available when viewing ion probability data
203. ialog box see Figure titled URL Chooser dialog box on page 7 10 14 5 Databanks PLGS or Database MASCOT This attribute specifies the protein or EST databank database that the mass spectrum data is to be searched against You can add PLGS databanks using the Databank Admin Tool see Organizing databanks with the Databank Admin tool on page 13 1 New Mascot databases can only be made available by your Mascot server administrator The list contains all available databanks databases Click the name of a databank or database to select it Tip It is advisable to specify a databank that contains the majority of protein sequences that could be in the sample data searched Rule Only one databank database can be searched at any one time any new selection replaces the existing selection Species PLGS or Taxonomy MASCOT These attributes are optional By default the entire databank database will be searched for matches to the data and all matches will be considered regardless of species or taxonomy PLGS databanks can be indexed according to species using the Databank Admin Tool see Organizing databanks with the Databank Admin tool on page 13 1 which allows searches using an indexed databank to be limited to one or more species Mascot taxonomies can only be changed by the Mascot server administrator To restrict the search to one or more species click the species in the list To select multiple species in the list u
204. id IP address or if it detects multiple IP addresses the Specify IP Address screen is displayed Rule If the installer detects a valid IP address this screen is not displayed Type the IP address of the network connection and then click Next If you cannot identify the IP address ask your system administrator for help in doing so On the Install as Services screen select whether you want to install as services e Yes The search engine and processor automatically run in the background when the PC is running Data on mapped drives cannot be processed or searched if the modules are run as services No default The search engine and processor only start when you start the ProteinLynx Browser Recommendation Select No if you are running PLGS on a single PC 1 4 Installing ProteinLynx Global SERVER 7 Click Next and then review the installation summary information If you wish to change any of the options click Back If you are ready to install click Install Tip Once the installation starts it can be stopped by clicking Cancel Once the installation is complete the Installation Successful dialog box is displayed Click Finish to close the Installer The ProteinLynx program group is now available ProteinLynx program group D ProteinLynx Browser h ProteinLynx 2 2 5 El Uninstall PLGS 2 2 5 Restoring backed up folders If you uninstalled a previous version of PLGS and backed up folders see Back
205. igh Low Automatic data curation rules Search Requirements for Requirements for Auto curation n i i engine OK assignment Maybe assignment PLGS Yes 95 probability 50 probability MASCOT Yes 95 identity Homology threshold threshold B 8 Scoring Schemes Implementing a plugin for ProteinLynx Global SERVER This section provides PLGS users with an overview of the plugin system used within the PLGS applications After reading this section you should understand the plugin architecture that exists within PLGS and also have an appreciation of how you can design and create your own custom plugins which can then be used within PLGS Contents Topic Page An introduction to the PLGS plugin C 2 Plugin architecture C 3 Use case the PLGS FileSystemPlugIn C 5 XML communication with the plugin implementation C 6 Adding a plugin to the PLGS application C 7 An example Executable plugin C 11 An example Java plugin C 13 Basic plugin Specific Queries C 16 Query tag definitions in the ProteinLynx DTD C 21 Plugin process exit codes C 26 UML Class Diagram for the PLGS plugin Architecture C 27 C 1 An introduction to the PLGS plugin C 2 A plugin can be thought of as a means to plug in to a system or application and allow for the transfer of data in to or out of that system Since PLGS 2 0 the PLGS applications have utilized plugins The default plugin used within PLGS is a
206. iii a Ne orien E 9 3 Tuporting a gel trom an OLB file serseri ruo a ndenanews 9 5 Importing agel from sample Dst snonsonssminnanimi fae 9 6 Replacing the sample in a well or spot eeessssssseseeeserererereresreesereeresserseeess 9 7 x Table of Contents PY OCESSINO data sadce aaa a aa a Aa E EAA a AERIS 9 8 Ganaa ean Pell Mata E E E AAE A T 9 9 AG el BG 05s odd cia dias A NE E P E E A 9 9 Viewing a summary of results for a Gel jcc scccssecasspivesseveesaisarsesaesecvessesesseveerays 9 9 Viewing sample annotation sssessesssssssessssssesssesssesssrosreosesoseereeeereessesereeree 9 10 10 Using Expression Analysis to compare and analyze sample groups E E A T A T T 10 1 Getting started with Expression Analysis cccccccccscccccceccesccseccescescees 10 2 Opening A proje eera avs A si end A EER 10 2 Experiment Analysis Design Manager sseessssssssssssssssssosssesssossoessosssoesoossoo 10 3 Experiment Atiributes sccccsccexsaccisniessmecenmnecadncieinmemianvdevesrcaterodiweeeainininawiceieioe 10 4 Select Grouping Method arsan enn SS 10 5 Manually Define Experiment Variables eseeeserrererserrsrrsserssrrssesssrssss 10 6 Manually Assien Samples To Groupe sissirsiissriisrsuci urei is erei 10 7 eg LL N E P EEE A A E E AE A E E E A EA eee E ETE 10 7 Pee Data UOU en aware asa 10 8 Guant tanon FAS ea A 10 8 Starting an Expression analysis ccccccccccccceeecceeceeeeeceeeseeceeeceeeseeseeeeeeeseness 10 9 Viewing
207. in Mass 14 8 Search Engine Type 14 5 Secondary Digest Reagent 14 10 Species 14 6 Taxonomy 14 6 Validate Results 14 12 Variable Modifications 14 11 databank searching real time 15 1 databanks 14 6 adding 13 3 archives 13 15 creating 13 3 deleting 13 13 editing 13 11 hyperlinks 4 4 real time searching 15 1 removing 13 13 restoring old 1 23 retrieving entries 6 30 search 14 3 14 13 database 14 6 data dependent acquisition See DDA DDA 15 1 15 8 A 22 A 23 DDA file setting up 15 10 De Novo Query 14 19 automated task 7 9 filter 7 11 sequencing parameters 14 21 De Novo Sequencing parameters 14 20 tool 14 1 14 19 validate results 14 22 deisotope peak detection 15 11 type 8 12 Index 5 Deisotoping and Centroiding attribute set 8 5 Deisotoping type attribute 8 12 Delete button 4 2 7 4 12 6 12 10 13 18 18 14 13 15 deleting archive files 13 14 databanks 13 13 digest reagents 12 10 modifier reagents 12 6 print templates 11 12 projects 3 6 sample 4 2 Delta Mass attribute 12 5 descriptions Databank Admin tool 13 2 Digest Reagent tool 12 7 Expression Analysis tool 10 2 Gel Manager 9 2 Print tool 11 2 processing parameters templates 8 2 Sample Manager tool 4 2 Design Manager Expression analysis 10 3 diagnostics displaying real time 15 15 showing 15 15 windows 15 15 dialog boxes Add Bookmark 2 11 Add Remove Tools 2 4 Automation Setup 2 18 2 24 Import Gel Spots 9 3 Installation
208. ing Searching parameters Continued Parameter Description Modifications Select and clear check boxes to set the fixed and variable modifications Real time status f f C To view the real time status click the Status icon The Real Time Status view is displayed Real Time Status page ProteinLynx Real Time Database Searching File RealTime Settings Help Real Time Status 0 MassLynx Idle RT 0 0 Raw File Proteins Excluded 0 MSMS Processing Submitted Queries 0 Protein Excluded Score Descripti Databank Searching Status 15 7 The following information is displayed Real Time Status parameters Parameter Description MassLynx Indicates whether MassLynx is acquiring data or idle RT The retention time during an acquisition Raw File The currently acquiring raw file Submitted The number of processed spectra that have been Queries submitted to the search engine Proteins Excluded The number of proteins that have been used to generate excluded lists In addition a table of results displays and updates details of the identified proteins including the protein name from the databank and whether that particular protein has been excluded Setting up a real time databank searching acquisition To set up a real time databank searching acquisition 1 Create a conventional DDA acquisition from MassLynx Setting up your DDA file on page 15 10
209. ing up the PLGS folders on page 1 3 you should restore them before starting PLGS To do this copy the backed up docs and root folders into the folder where you installed PLGS If you backed up databanks they must be re added to PLGS For details on how to do this see Adding databanks on page 13 3 Running PLGS on Windows in a client server environment To run PLGS in a client server environment you need to start these PLGS modules on each computer e Microkernel Search engine Processor All of these modules are started automatically when you start the ProteinLynx browser on that computer To start the PLGS browser 1 Click Start gt All Programs gt ProteinLynx gt ProteinLynx Browser 1 5 Running PLGS on Windows on a single PC To start ProteinLynx Global SERVER click Start gt All Programs gt ProteinLynx gt ProteinLynx Browser Starting modules manually and troubleshooting problems All of the modules on a computer are started automatically when you start the ProteinLynx browser Nevertheless you might wish to start the individual modules separately To start PLGS modules manually 1 Navigate to the PLGS installation directory and then to the bin subdirectory 2 Start the module by double clicking in Windows or by typing its name at the command prompt ProcessorEngine bat to start the processor SearchEngine bat to start the search engine PLmicrokernel exe to start the microkernel If
210. ing your data To enable these filters click Use Additional Filter Settings Additional filters Filter Effect Display all items with the following Only those clusters or proteins OK level s marked with the selected status see To curate organize your data on page 10 11 are included Remove all proteins with a score less Only proteins with a score higher than than the value entered are included 10 15 Additional filters Filter Remove all EMRTs with an average mass error PPM less than Effect Only EMRTs with an average mass error the root mean square calculated in parts per million greater than the value entered are included Average mass errors are typically very small Remove all EMRTs with a percentage CV in retention time greater than Only EMRTs with a coefficient of variation in retention time that is smaller than the value entered are included Remove all EMRTs with a percentage CV in intensity greater than Only EMRTs with a coefficient of variation in intensity that is smaller than the value entered are included Importing workflows Import workflows to apply the protein identification results of one or more databank searches to your EMRT results table To import workflows 1 Click the Import Workflows button S 2 Inthe Select Workflows dialog box select the boxes on the rows relating to the workflows you wish to import 3 Click OK
211. input from stdin and writes it to file include lt fstream gt include lt iostream gt include lt string gt using namespace std int main int argc char argv ofstream out file to write input to out open helloplugin1 txt 1os app ensure file has opened if lout cerr lt lt HelloPlugin1 ERROR OPENING helloplugin1 txt lt lt endl return 3 while cin cin getline c out lt lt c out lt lt end close file out close return SUCCESS exit code return 0 C 12 Implementing a plugin for ProteinLynx Global SERVER An example Java plugin All Java class plugins must implement this interface to become compatible with PLGS The PlugInImp interface has 2 methods Processes the input read from input stream writing output to output stream Error output is directed to error stream param inputStream the input stream param outputStream the output stream param errorStream the error stream return 0 for success exception java lang exception when the processing cannot continue due to an error int process java io InputStream inputStream java io OutputStream outputStream java io OutputStream errorStream throws java lang Exception Sets properties for this PlugInImp Called immediately after the PlugInImp is instantiated param properties the properties for this PlugInImp exception implementations should throw an I
212. int data for a whole project clear the box For a template to print data for specific workflows only select the box Rule The Tabular Data option is only available if you have set up the printing preferences to enable quick table pages See Printing tab on page 2 16 for details 5 Click Next and then select the ways that you want information to be grouped Tip The selections that you make are displayed in the Results section of the template navigator tree in the same order as they are displayed in these screens 6 Click Next and then select the data sets to be displayed You can change the order of the data sets by using the up and down arrows 7 Click Finish Results e The template details are shown in the Print Tool view in the browser The Table Setup selections are chained in the Results section of the template navigator tree 11 14 Creating print templates and printing project data Print Tool Table Setup display in the navigator tree of a template Print Tool TestProjectTemplate i HeaderiFooter Header Footer ntroduction D D 7 pm Project content Locations content Hits content Proteins content L f Peptides content L Summary You can still add content to the Results section after the template has been created 8 Click to save the template Adding content to the results nodes You can add content for the grouping and data s
213. inutes m Acquisition lonization Mode Source ES Polarity Positive Negative Analyser Mode VMode W Mode Dynamic Range Nomal f Extended Recommendation When using BEH 75um columns a start time of 10 minutes and an end time of 75 minutes is suggested for a 60 minute LC gradient 5 Click the Expression tab 6 Enter the low collision energy value and the ramp for the elevated collision energy 16 4 Using MSF for qualitative proteomics Expression tab Expression Criteria Ramp High Energy from f15 to 40 volts Low Energy 4 volts The ramp for High Energy is typically set to 15 eV to 40 eV Click the TOF MS tab and enter the values as shown below TOF MS tab Acquisition TOF MS Expression Cone Voltage LockMass MS Survey Da Range Acquire MS Survey over the range Start Da End 1900 Da MS Survey Scanning Conditions Scan Time 15 seconds Data Format Tip The mass range over which you wish to acquire data is typically 50 m z to 1990 m z Recommendation When using BEH 75um columns a scan time of 0 6 seconds is suggested 16 5 8 Click the LockMass tab and then enter the values as shown below Rule The Reference Scan section of this tab is available only if the Tune window gt Mode gt LockSpray option is checked Mass accuracy and therefore Lock Spray is an integral part of the Expression approach to data acquisition LockMass tab Acquisition TOF MS Expression
214. is method is invoked with a status code PlugInHandlers can be implemented for specific tasks although some generic implementations can prove useful an OutputStreamPlugInHandler for example C 3 C 4 Currently there are two plugin implementations provided with PLGS Executable and Java class implementations Executable plugins or ExecPlugIns extend the plugin interface to allow executables to be used to import and export data into and out of PLGS Java class plugins extend the plugin interface to allow classes which implement an additional interface called the PlugInImp interface to import and export data into and out of PLGS PlugInImp classes simply process input from the plugin through an input stream process output from the plugin through an output stream and process error messages from the plugin through an error stream A UML class diagram of this plugin architecture can be found in UML Class Diagram for the PLGS plugin Architecture on page C 27 The client of a plugin is the item or entity that calls and runs that plugin The dialogue required between a client and a plugin is particularly simple all input is provided by the client and then the input stream is closed the client of the plugin then waits for output or the termination of the plugin process All plugins have an associated PlugInHandler that will handle plugin events such as the start of the plugin process handling output from the plugin handling errors form
215. is updated with the information from PLGS Data can now be acquired in the normal way A 10 Quick Start Tutorials Example MassLynx sample list wi MassLynx PLGS2Training untitled File View Run Help 2 0 B H D 1 UD A shou youve Estus Queue Is Empty E Instrument Spectrum Chromatogram Map Edity Samplesv z Bottle File Name File Text MS File Inlet File 5 o 1 Al Sample_0 digestLMC exp Default A 2 A2 Sample_1 digestLMC exp Default Inlet Method 3 A3 Sample_2 digestLMC exp Default 3 4 A4 Sample_3 digestLMC exp Default F 5 IAS Sample_4 digestLMC exp Default x amp 6 A6 Sample_5 digestLMC exp Default S MS Method m IA Sample_6 digestLMC exp Default S 8 A8 Sample_ digestLMC exp Default N Gv 3 a3 Sample_8 digestLMC exp Default 10 A10 Sample_9 digestLMC exp Default g MS Tune WAN Sample_10 digestLMC exp Default o 12 12 Sample_11 digestLMC exp Default Z D 13 B Sample_12 digestLMC exp Default 3 Edit Shutdown or Startup 14 B2 Sample_13 digestLMC exp Default 5 15 B3 Sample_14 digestLMC exp Default W 16 B 4 Sample_15 digestLMC exp Default VW IBS Sample_16 digestLMC exp Default R shutdown 18 B 6 Sample_17 digestLM C exp Default D 19 B Sample_18 digestLMC exp Default s 20 B8 Sample_19 digestLMC exp Default E gt 21 B9 Sample_20 digestLMC exp Default Startup 22 B10 Sample_21 digestLMC exp Defa
216. ist Modifier Tool existing modifier reagents lists Modifier Tool Amino Acid Modifier Reagents Carbamidomethyi C Carbamyi K 42 01056 K See Reagent attributes on page 12 4 for details of the attributes and values Rule The values of supplied modifier reagents gray text cannot be edited 12 3 Adding and editing custom modifier reagents To add or edit a custom modifier reagent l To add a reagent click the New button E on the toolbar or click File gt New lt To edit an existing custom reagent click the reagent in the list Tip Existing custom modifier reagents are shown in black text in the list For both actions a panel and text box are generated which enable defining or editing of the values for each attribute Adding a new modifier reagent Edit Modifier Reagent Attribute Modifier Type Quantitation Reagent No Delta Mass Da 0 0 Applies To Fragments Name Enter the name of the modifier reagent Name Rule Only user defined modifier reagents can be edited you cannot edit the supplied modifier reagents 2 Click a row in the panel to update the value of the attribute You can amend the values for the following attributes Reagent attributes Attribute Description Type a unique descriptive name this name is used throughout the system The supplied reagents use the format lt reagent name gt
217. k Matching MALDI PSD MX only Chromatogram Electrospray MS and Electrospray High Low only Restriction Some attributes in the attribute panels are disabled and these cannot be edited Some of these grayed out attributes have default values that are used by the processor MALDI PSD MX For the Noise Reduction and Deisotoping and Centroiding attributes two template panels MALDI MS PSD MX are displayed which have related attributes The panels labeled MALDI MS represent the processing to apply to MALDI MS data the panels labeled PSD MX represent the processing to apply to PSD MX data MALDI Q Tof MSMS For the Noise Reduction and Deisotoping and Centroiding attributes two template panels MALDI Survey MSMS are displayed which have related attributes The panels labeled MALDI Survey represent the processing to apply to survey data the panels labeled MSMS represent the processing to apply to MSMS data 8 5 Electrospray DDA QTOF MSMS For each attribute two template panels Electrospray Survey MSMS are displayed which have related attributes The panels labeled Electrospray Survey in each attribute represent the processing to apply to survey data the panels labeled MSMS represent the processing to apply to MSMS data Mass Accuracy attributes Not all attributes are available for all panels check the Applies to column in the table below to see whether the attribute listed relates to the panel you are configuring
218. k of Red Hat Inc ICAT is a trademark of the University of Washington iTRAQ is a trademark of Applera Corporation Other trademarks or registered trademarks are the sole property of their respective owners QUALITY 1 MANAGEMENT Intended use ProteinLynx Global SERVER can be used as a research tool to deliver qualitative protein identification and relative quantification It is not for use in diagnostic procedures Customer comments Please contact us if you have questions suggestions for improvements or find errors in this document Your comments will help us improve the quality accuracy and organization of our documentation You can reach us at tech_comm waters com Table of Contents 1 Installing ProteinLynx Global SERVER sssssssseeeeeeeeeeeeees 1 1 Typical client server installation esssesssssssesssessosssosssossosssoessosseseseosseceesoseos 1 2 Installing PLGS on Windows 1itiireririsirsinresisrsinrsersenrenretnrsnreeretnr 1 3 Backing up the PLGS folders cccccccccccccccccecceccceesecceecccecceeceseceeseeseeseeens 1 3 Beckie tip dataan ES seralar e T S 1 3 Uninatalhing PLGS in Windows ss cssicaisciisraiseisiecieisicstientaisiamenaies 1 3 Installing PLGS on WindOWSacusiecnceisacisnarna aae 1 4 Restore backed up folders sessriru rE EA 1 5 Running PLGS on Windows in a client server environMent c ccceeeee 1 5 Running PLGS on Windows on a single PC sssssssesesssssssssr
219. kflow node is displayed You will attach search methods queries to this node By default the title of the template is the current date and time which is shown in the Editor panel If desired type a new title in the Title text box Workflow Designer workflow node Workflow Designer Workflow Attribute Value Title Workflow created on Thu Jul Workflow Node Title Enter a title for the workflow template Title AACE COAL Jul 29 2004 at 12 17 21 3 Right click the workflow node and then click Add 4 If this is the first time that you have attached a search method click Databank Search For some search types Databank Search is the only available option The attributes displayed in the attribute table of the Editor panel vary slightly depending on the type of search engine PLGS or MASCOT Rule MASCOT is only available for selection if you specify a Mascot search engine in the browser Preferences dialog box See Search Engine tab on page 2 5 7 6 Defining templates for searching with Workflow Designer Databank Search attributes PLGS search engine Workflow Designer Search Engine Type Databank Search Query L atabank Search Query m Workflow Fragment lon Search rf lt PLGS Databanks Species Peptide Tolerance Fragment Tolerance Estimated Calibration Error 0 005 Da Molecular Weight Range Oto 200000 Da pl Range Minimum Peptides to Match
220. kflow report on page 11 10 or export the data to a csv or html file type Recommendation It is reeommended that you preview the report The Edit Limits dialog box enables you to override the limiting options for the results that are set in the template see Limiting results on page 11 17 However the settings in this dialog box are not saved in the template 4 After selecting an option click Finish 11 9 Previewing a Workflow report i Page Preview WorkflowProteins B E E v Results coverage mae 2 MSMS spnctram dadare aplcatsa ant ther evtance ant data maxa ipinya peptic are arta by cicraanrg SOONE Nes Author rou Date of Priima Dac 27 304 Wertesataters ALL OKOV AI NQOPLAK ALL OKOV I AI NQOPLGK ETU The toolbar has various functions Print preview toolbar functions Function Description Print Print the workflow from this screen Import Import another workflow to be previewed printed or exported Export Export the workflow results to a csv or html file 11 10 Creating print templates and printing project data Print preview toolbar functions Continued Function Description Refresh Refresh the preview Toggle Preview pages horizontally across the display grid Zoom Increase or decrease the scale of the view range 25 to 2
221. kflow templates cccicscsscsscsessasasaccessansoscasvencsdssvadeavsavervidenvsaveuvsvanver 7 9 Opening workflow templates c cccccssccssecssecscccscccssccsccceccesccseeeeseeseeesseass 7 10 BOMBS 5 oicecs wa ceaios E E EEE O E E E E EER 7 11 Pam Mod EN esras E a gee ane uadannnnaienaainencinetan 7 11 De De TC aa A 7 11 8 Creating custom processing parameters e sesesssecseecsssssseesseeececeeeeeooo 8 1 Getting started with the Data Preparation tool sesssssseseseoseossecoseoseosoeeoo 8 2 Attribute sets for data preparation ccccccccceccecccsecceccesecsecceseeseccesecseeseceess 8 5 ee PAO M erasi a E ETES 8 5 MALDI Ge Far MSME renjar nii A E 8 5 Electrospray DDA QTOF MSMS n cicsstvidnormitivon a i 8 6 Moss Accuraat AtA DUIO ern S SS 8 6 Noise Reduction abbr iiss sacescincscnsssardircasarnrisbinrenrsincsisemeiercrasmsieerns 8 9 Deisotoping and Centroiding attributes ssscsccsssseesesssseesessssesseseceeees 8 12 Peak Matching attribnt Snosccncnaccuneni 8 15 C hromatosram AINUT spiicata e a ani 8 15 9 Viewing and processing gel data with Gel Manager 006 9 1 Getting started with Gel Manager ssessssssssssccsccessssssccccesesssscccesessssecccesesseseeoe 9 2 Adding and importing data cccccccccccscrsceccccccccccecccecceccceccceccseccesecscccececeeces 9 3 Adding a new gel without an image cccccsssssscssssssessssssesssessnsesesteneesees 9 3 Uy ceteris gel spote sorsrinrinrn
222. kground A value of 0 corresponds to a flat threshold and 1 is a sloping straight line For typical data a value of around 5 will be sufficient Perform Smoothing Smoothing Type All All Whether to perform smoothing Smoothing removes rapid variations in intensity and can improve peak detection results The smoothing method to use Savitzky Golay smoothing preserves line width better than Mean smoothing Smoothing Iterations All The number of times that the smoothing should be performed Smoothing Window All The half width of the smoothing window in channels Combine Options MALDI MS not MALDI PSD MX MALDI Survey The method of combining scans The reference external lock mass scans are never combined with sample scans The setting of this attribute will affect whether other attributes are available Recommendation The recommended setting is All Scans to Combine MALDI MS PSD MX MALDI Survey The number of scans to combine This option is only available when Combine Options is set to User input 8 10 Creating custom processing parameters Noise Reduction attributes Continued Attribute Applies to Description Low Mass Threshold MALDI MS The low mass threshold Only PSD MX data above this threshold is MALDI Survey used to determine which scans to combine This option is only available when Combine Options is set to Auto select In
223. ks are considered to be signal Threshold Precursor The percentage of the precursor mass for the tolerance Matching Window of the precursor masses Fragment The tolerance in parts per million ppm of the fragment Matching Window masses Report Selected Yes Monoisotopic fragments are reported Monoisotopic Cleared No Average fragment masses are reported Fragment Masses Calibration File Default None File Opens the File Chooser dialog box Navigate and choose file The file path and name are displayed in the box Clear Selects None Chromatogram attributes The Chromatogram attributes are available for the Electrospray MS and Electrospray High Low panels Chromatogram attributes Attribute Description Minimum Peak The duration in scans or time for which the threshold Width criterion must be met for a peak to be reported Expected Peak The expected peak duration full width half maximum Width This is used to help decide when ions start and stop eluting 8 15 Chromatogram attributes Continued Attribute Description Peak Width Units The unit by which peak width should be measured Automatic When automatic thresholding is used the deisotoping Thresholds algorithm attempts to choose a sensible threshold for every spectrum that it is given Although processing the data in this way should give reasonable results experienced users might wish to
224. l digest using a second reagent is carried out on peptides produced by the first digest Select a reagent from the list as for Primary Digest Reagent PLGS or Enzyme MASCOT on page 14 9 Missed Cleavages This attribute is optional as a default number is supplied This attribute specifies the maximum number of missed cleavages permitted when generating the set of peptides produced by a theoretical protein digest The value is applied to the primary and secondary digest reagents except where a non specific reagent or None is selected Fixed Modifications This attribute is optional By default no Fixed Modifications will be applied to the peptides produced by the digests The list contains all available modifier reagents 14 10 Query Tools To specify a modification that should always be applied to peptides produced by the digests click the desired reagent in the list To select multiple reagents in the list use Shift click to select consecutive reagents or Ctrl click to select non consecutive reagents For a PLGS search to create additional modifiers to the existing list use the Modifier Tool see Getting Started with the Modifier tool on page 12 2 For Mascot searches see your Mascot server administrator Variable Modifications This attribute is optional By default no variable modifications are applied to the peptides produced by the digest You can apply any number of variable modifications to the peptides gener
225. le Workflow created on Thu Jul Workflow node Title Enter a title for the workflow template Gel Manager n Workflow created on Thu Jul 29 2004 at 12 17 21 Container Manager Workflow template Expression Analysis Databank Search Editor panel Desktop panel lo AutoMod Analysis j nO006 Workflow Designer 7 3 Workflow Designer toolbar The following table describes the buttons on the Workflow Designer toolbar and their corresponding menu bar options Workflow Designer toolbar options Button Menu Bar Option Description E File gt New Adds a new workflow template to the desktop i panel File gt Open Opens a previously saved workflow template File gt Open URL Opens the URL chooser dialog box see Figure titled URL Chooser dialog box on page 7 10 to enable you to specify a remote source that contains a workflow template File gt Remove a Removes the selected workflow template nee internal frame and discards all changes File gt Save Saves the selected template As File gt Save As Prompts for a name and saves the selected template File gt Print Prints the workflow template and all its automation parameters Edit gt Add Opens a list which shows all the available automation tasks that can be added to the template Edit gt Cut Removes the selected node and all o
226. lick Open Expression Analysis Result The Design Manager opens at the section that needs your attention next 10 3 Expression Analysis Design Manager File Edit View Windows Options Tools B amp m amp amp E Projects CSF_225Processing X pression Ana CSF_225Processing 4 Expression Analyses qh 132_407 periment ana y ce vill2 Expression Analysis Result EMRT Cluster Data Experiment Name ExpressionAnalysis 1 EAN Protein Table Ba expressionAnalysis 1 Description Processing Parameters Templates D Workflow EE i Apy A A a a i a a 4 Experiment Attributes Each expression enalysis experiment has 2 attributes that must be set A name for the analysis and also a description ofthe analysis These attributes help to give each analysis a readable ID and also provide a means to provide a short description of what is in each analysis 4 gt No jobs Note the following details which apply to the Design Manager s seven sections e Ared title indicates the section that needs completing next A blue title indicates that the section is active but that another section should be completed first To apply the values that you specified for a section and progress to the next step click Apply To see or edit the values of another section click the arrow at the right of the section heading Click the arrow again to hide the section Ex
227. llegalArgumentException if necessary properties are absent or invalid void setProperties java util Properties properties The following is the source code for an example Java class plugin called MirrorPlugIn java This plugin will print out the input it receives to the System out Notice how this class implements the PlugInImp interface Add this plugin in PLGS to see how it works In order for the MirrorPlugIn to become available you must compile it and place the MirrorPlugIn class into a jar file with the PlugInImp class which can be found in the proteinprobe jar file in the PLGS installation folder called jars Created on 26 Sep 2003 package MirrorPlugIn import java io InputStream import java io InputStreamReader import java io OutputStream import java util Properties import uk co micromass plugin PlugInImp author NEESONK To change the template for this generated type comment go to Window amp egt Preferences amp gt Java amp get Code Generation amp gt Code and Comments public class MirrorPlugIn implements PlugInImp This is the main process method of all Java plugins oe public int process InputStream inputStream OutputStream outputStream OutputStream errorStream throws Exception C 14 Implementing a plugin for ProteinLynx Global SERVER System out println The MirrorPlugIn has been called InputStreamReader reader new InputStreamReader inputSt
228. low templates on page 7 10 Check that each databank field contains a databank If a databank is not shown the previously set databank is not present on your currently selected server This is an issue when opening up older workflows created with a previous version of PLGS 5 33 5 34 Specifying samples vials and plates with Container Manager Viewing results in the Results Browser Following acquisition and processing the data can be viewed in the workflow results browser A separate results browser is opened for each set of results This section describes how to view results and use the results browser Contents Topic Page Viewing results 6 2 Results browser 6 3 Protein Workpad 6 27 Exclude Masses Workpad 6 31 6 1 Viewing results The results browser for each set of results can be opened in several ways Each set of results is listed in a Results Summary table To view results in the results browser either Click a well or spot on a plate right click and then click View Results or Double click the workflow results node in the Container Manager navigator tree or Click anywhere in a row of the Results Summary table To view a larger Results Summary table Hide the tool tray by clicking the arrow 4 on the blue splitter bar between the tool tray and the display area of the main PLGS window Hide the navigator tree panel by clicking View gt Maximise Desktop Results Summary table EE A A
229. lt The replicates or peptides for the selected cluster or protein are displayed To export your data Tip If there are many results you might wish to filter the results see page 10 13 before exporting them K 1 Click the Export Data button 2 Inthe Export Data dialog box select the boxes beside the columns you want to export and clear the boxes beside the columns you do not want to export 10 12 Using Expression Analysis to compare and analyze sample groups 3 Click OK 4 Type a name for the export file and then click Save Result A tab delimited file is created with the specified name If there are many results it can take a few moments for the export file to be created To print your data Tip If there are many results you might wish to filter the results see page 10 13 before printing them c 1 Click the Print Data button The Print Wizard see Using print wizards on page 11 3 is displayed 2 Follow the on screen instructions in the Print Wizard clicking Next to progress from one step to the next and Finish to print To include exclude all clusters To include all clusters click a To exclude all clusters click a Rule Only one of these buttons is displayed at any one time If the Include All button is clicked the Exclude All button is then displayed If the Exclude All button is clicked the Include All button is then displayed Protein table The Protein table is similar to the EMRT table
230. ly if you want to export results from a container directly to this PlugIn bypassing the FileSystemPlugIn and any other third party PlugIns 2 25 Attributes Executable Plugin Attribute Executable Description Click E to browse for the location of the executable or type the full path to the executable Working Directory Click E to browse for the location of the directory to which you want the PlugIn to write its files or type the full path to the directory Arguments Type the list of command line arguments required by the PlugIn Export Selected Results from Container Select this to export selected results from a container directly to the PlugIn Default Cleared Save Projects from Browser and PeptideAuto Select this to execute the PlugIn whenever projects are updated by the browser or PeptideAuto Default Selected Attributes Java Class Plugin Attribute Description PlugIn Name Optional Required only if you want to export results from a container directly to this PlugIn bypassing the FileSystemPlugIn and any other third party PlugIns Class Path Click to browse for the location of the jar file or class or type the full path to the jar file or class 2 26 Setting up ProteinLynx Global SERVER Attributes Java Class PlugIn Continued Attribute Description Classes Implementing When the plugin s jar or class file h
231. m the Container Manager navigator tree or a results window Note The speed of rendering depends on the amount of data being applied to the template and the specification of the computer 11 2 Creating print templates and printing project data Using print wizards To print project or workflow data you use the project or workflow print wizards You can print project data from the navigator tree of any tool that shows the project name However you can only print workflow results from the Container Manager navigator tree or a results window Project print wizard To use the project print wizard 1 Inthe navigator tree of any tool click the project name and then right click Project print wizard pop up menu in navigator tree Container Manager m test 588 vials B R Microtitre PI Foe Workflow Results 20 p late on gt Gi e Vite md wile 91293 Carbamoyl niil 23495 Hypothetica illc QIQUN Toll like rec nlll P55123 Leukotoxin nile Q12495 Chromatin wile Q97VWWHO DNA doubl niil P93844 Phospholip lt nille Q03410 Synaptoner illc PO1029 Complemer AA 10 42 2 50 CAExpres AMC Hans Aerts ci gt c2 D1 gt D2 2 Plate2 i EA Target Plates Processing Parameters Template 77063 02 a max 2 Click Print 3 Select either default templates or user defined templates and then click N
232. m the selected protein or EST Hold down the left mouse button to drag and drop the protein or EST onto another component Selecting peptides from the table To select a peptide from the table click the relevant row The MS spectrum display highlights the peak mass that is matched to the peptide The MSMS spectrum display shows the fragmentation spectrum for the peak mass that is matched to the peptide and annotates the spectrum with the peptide fragmentation data Hold down the left mouse button to drag and drop the peptide onto another component Resubmitting the search The spectrum data with some peaks excluded can be resubmitted for a search The resubmitted search uses the same query parameters as the search that produced the original set of results Results browser 6 15 To resubmit the unmatched peaks from the spectrum for a search that is excluding all peaks already matched to a peptide right click either the protein or peptide table and then click Exclude Re submit gt Resubmit with Current Exclude List lt To resubmit all peaks not specifically excluded from the spectrum right click either the protein or peptide table and then click Exclude Re submit gt Resubmit Excluding Current Protein Peaks can be excluded from resubmitted searches using the Exclude Masses Workpad described in Exclude Masses Workpad on page 6 31 Note Masses selected for exclusion are usually theoretical masses which can differ
233. mages 1 Right click the Template Details node and then click Insert gt Image 2 In the dialog box under the navigator tree click the Image page and then click Browse Print Tool adding images YA Proteinlynx Browser File Edit Insert View Options Tools Ban 6 amp 8 2 amp amp Print Tool I SampleProjectTemplate m HeadenFooter 4 C Header Sample Manager rs aih E Waters For Complete Confidence Footer c i LE Field D Introduction Gel Manager C Template Details PA ace n amm Image element D Results C Project content Container Manager Locations content C Hits content E Proteins content L A Peptides conte L Summary E AE 8 Expression Analysis Image Selection dialog box 5 e 6 e m s s alo Databank Search S A YA Choose an Image AutoMod Analysis Look In a Print_Templates C watersHeaderjpg WatersmarginBottomRightjpg De lovo Sequencing File location BLAST Searching Print TemplatesiwatersHeader pg E L nome A Data Preparation JPEG Images D 5 MAT Print Tool 3 In the dialog box browse to an image file click the file and then click Open 4 Use the settings in the Dimensions tab to change the position and dimensions of the
234. mass value Items that represent a peptide have only one associated mass the molecular weight of the peptide Items that represent a hit protein or EST have multiple associated masses Each associated mass is the molecular weight of a peptide that is a match to the protein or translated EST sequence Items that represent a digested protein or EST have multiple associated masses that represent the molecular weights of peptides but in this case the peptides are the fragments produced by the simulated protein digest Results browser 6 35 6 36 Viewing results in the Results Browser Defining templates for searching with Workflow Designer The Workflow Designer enables you to define a template that can be used to perform an automated databank search of samples in the Container Manager and Gel Manager Contents Topic Page What is Workflow Designer 7 2 Creating a workflow template 7 5 Filters 7 11 What is Workflow Designer The Workflow Designer enables you to define a template that can be used to perform an automated databank search of samples in the Container Manager and Gel Manager To search MSMS MS or PSD data you can use these search types e PMF Peptide Mass Fingerprint gt PMF Fragment Ion Search Fragment Ion Search To search Expression MS data use these search types Electrospray MS for low energy MS only Electrospray High Low For each of these you can use the Databank S
235. microkernel and browser Starting the browser automatically starts the other components Each component can be started manually if required The browser enables you to add new databanks to the server or view help about the system Before running PLGS ensure that you are logged on with root permissions To start the PLGS system 1 To start PLGS go to the directory lt PLGS install location gt bin 2 Type ProteinLynxBrowser to start the browser Restriction Only the Databank Admin Tool and the online Help are available in the UNIX PLGS browser If so configured processing and searching can be run on a UNIX machine from a remote Windows PLGS browser Starting modules manually and troubleshooting problems All of the modules are started automatically when you start the ProteinLynx browser on the computer Nevertheless you might wish to start the individual modules separately however Before running PLGS ensure that you are logged on with root permissions To start modules manually 1 Go to the directory lt PLGS install location gt bin 2 Start the modules by typing the following at the command prompt SearchEngine to start the search engine PLmicrokernel to start the microkernel ProcessorEngine to start the processor If you start the modules automatically by starting the ProteinLynx browser log files are generated by the software These log files can help you to solve operational problems and
236. ministrator and the users running PLGS are not Make Blastable If this option is set to TRUE the necessary index files are created and the databank will be available for BLAST searching by using the BLAST Searching tool Index For PepGrab If this option is set to TRUE the necessary index files are created and the databank will be available for PepGrab searching via the PepGrab function Load into Memory Loading a databank into memory increases the speed at which that databank can be searched by the Databank Search Tool Ensure sufficient RAM is available Select True or False as required Tip Databank searches can fail if very large disk based databanks are used If a failure occurs try loading the databank into memory 13 6 Organizing databanks with the Databank Admin tool Databank attributes Continued Attribute Species for Indexing Description When a databank has been indexed by a species a Databank Search restricted to that species can be performed using the Databank Search Tool Any number of species for indexing can be selected for indexing Each species for which the databank has been indexed will appear in the Databank Search Tool species list for that databank Select any combination of species To select more than one species hold down the Ctrl key while clicking the required list elements Management Options If further management options are required set this
237. modifications Each peptide in each protein in the database is given a prior probability the weight of which is determined by its end amino acid the number of missed cleavages it contains and number of variable modifications it has undergone B 3 MALDI scoring PMF PMF fragment ion searches The scoring scheme implemented in PLGS 2 2 5 for MALDI data gives a quantitative answer to the question Which single protein best accounts for the data given some initial assumptions The data consists of a set of mass intensity pairs and their associated uncertainties representing the mono isotopic mass and intensity of every peak in the processed data above 900 Da All matches inside the user set tolerance are recorded and ranked according to the scoring scheme The reported score indicating how much of the total probability a protein has is given by Protein Score In Probability of Protein GIVEN Data AND Initial Assumptions Probability of Protein GIVEN Initial Assumption If there are N proteins in the databank each protein in a databank has a prior probability of 1 N Therefore the maximum possible score is In N and the minimum possible top score is zero when the data provides no information relative to the databank The posterior probability of Protein GIVEN Data AND Initial Assumptions is also presented as a percentage B 4 Scoring Schemes MSMS scoring fragment ion searches The
238. mote address and checks that it can be accessed This can take a few seconds Download Compression Type Rule This option is only available if the Periodically Download attribute is set to True This field relates to the periodic download of remote files Databank flat files available at public sites are often stored in a compressed form to save space The Databank Admin tool will automatically decompress several types of compressed file including z Z zip and 2z compression types If known you can specify the compression type of the remote file If the field is left as Unknown then the system decides the compression type Download Renew Period Rule This option is only available if the Periodically Download attribute is set to True Enter the number of days after which a new databank flat file will be downloaded Download processing will only take place between the Start and End times The default period between downloads is 30 days In the text box type a whole number greater than zero 13 8 Organizing databanks with the Databank Admin tool Databank attributes Continued Attribute Periodically Update Description Rule This option is only available if the Management Options attribute is set to True To periodically update the databank from a remote location using interim update files set this option to True Some providers of databanks supply interim update files which contain
239. mple List to MassLynx Export to MassLynx dialog box P7 Export to MassLynx Project QTOFMSMSPRO sy Method File atotexp oy Inlet File Default v Tune File latoti ooo isS File Name a Tof MSMS Data Name sample_ i Number of samples to be included in list 1 Once the list has been produced the processing parameters for hese samples may not be changed Samples included in a previous list will not be included in this list nless data acquisition was cancelled before spectra were obtained 2 Specify the MassLynx project from which the data is to be acquired 3 If more than one MS Method is stored in the MassLynx project use the drop down list to specify the correct file Tips The File name can be the same as the target plate name The MS Data name can be changed to any text such as digest_0 adh_0 4 Click Export 5 In MassLynx click File gt Import Worksheet 6 The file created by PLGS is stored in the MassLynx project Browse to the file and then click Open Result The MassLynx sample list is updated with the information from PLGS Data can now be acquired in the normal way A 20 Quick Start Tutorials Acquiring data As the instrument begins to acquire data chromatograms are recorded MS data MSMS data and lockmass correction data is also obtained When the instrument switches into MSMS mode the ions selected for MSMS ar
240. mpute their relative abundance Indicate whether they are upregulated or downregulated A wizard simplifies the complex of setting up an Expression analysis experiment The wizard takes you through the process of specifying your samples and settings Note The Expression software can be used as part of the optional Waters Protein Expression System The Waters Protein Expression System provides a number of additional features including label free analysis For more information refer to the Waters Protein Expression System Operator s Guide To open the Expression Analysis tool click the Expression Analysis icon Opening a project Before creating an Expression analysis you must create a project see Creating a new project on page 3 2 To open a project that you have created click the drop down list in the toolbar and then clicking the project you wish to open 10 2 Using Expression Analysis to compare and analyze sample groups Experiment Analysis Design Manager The Experiment Analysis Design Manager leads you through the creation of an Expression experiment To create a new Expression experiment 1 Click Expression Analyses 2 Right click and then click New Expression Analysis Result A new Expression analysis is created in the tree and the Design Manager opens at the first stage Experiment Attributes To open an existing Expression experiment 1 Click the name of the experiment 2 Right click and then c
241. n A A RAAEN 1 19 Starting modules manually and troubleshooting problems 06 1 19 Installation troubleshooting on UNIX erorar ie 1 20 Installer startup propilene nnreniimnndieniin aii 1 20 Mierokernel TR serran EA T EIRE 1 20 pearch engine TUPI AG co crssitd star stance arate vonnnronvnad R A iS 1 21 Large databank gt 2 GB problems sac ncncuinsndieiiiesiaianiis 1 21 Databank and BLAST searching problems cccccccscssseeseeesteeeeeeees 1 21 Restoring old databanks sissscsccscssecscssccsssesecassassscsswetccesseseseecostveesvesessusccassesase 1 23 Setting the number Of processors ssssccccssssssssscsscccsssssssssssscssssssssssssees 1 24 DDA data pro essing eiicccssscisissivncsevissavsnvassurssiuavovsavesvsnededaidessndantassnsarersnaeics 1 24 Expression data DECE CHI ok ceored idiots ne ees 1 25 Parabank cerr hii oie tains as 1 25 2 Setting up ProteinLynx Global SERVER ssesessesosssosssssseeeseeseceoeeeeose 2 1 Protein Lynx Dv OWSEY csser annees aE A ES EE EAE E aaas 2 2 LOLIT a wads Set te aah pe ee ea ts a iad epider tcayeiuen 2 3 Adding and removing tOolSuses seniem n a anaes 2 4 Changing preferentes sciisssccsssctsasscecivicsavsiasanecsevasiaavinsepcansnacddeesenceaecsaaicssensaaniivs 2 5 Search Engine tabccnsinaniscrni innean A RS AE RRENEN 2 5 Adde a search COSINE esting aie E week 2 6 Modiying a search nane sosro s S 2 7 Removing asearech enaNe cccsancrcanitemcininincae i aeenieamieee 2 8 Procon aiie
242. n click OK Deleting items from the excluded list To delete an item from the Excluded Masses Workpad 1 Click the item in the Excluded Masses Workpad 2 Right click and then click Delete Exclude Tip To select multiple items press Shift or Ctrl while clicking Running a simulated digest for a protein To add a new item that represents a digested protein or EST to the Exclude Masses Workpad 1 Click a protein or EST in the Excluded Masses Workpad and then right click 2 Click Use Reagent 3 Click a digest reagent in the list Result A new item representing the digested protein or EST is added to the list Results browser 6 33 Exclude Masses Workpad with digested protein added RIDH_KLEAE Slymotrypsin Viewing the masses associated with an excluded item To view the mass values associated with an item in the Exclude Masses Workpad 1 Click an item in the Exclude Masses Workpad and then right click 2 Click View Exclude Masses Result A separate window is displayed showing a list of the mass values Masses to Exclude window JaMasses to Exclude xi DHB_ANAPL 7I 1275 682 y 1291 677 6 34 Viewing results in the Results Browser 3 Select a check box to exclude that specific mass from resubmitted searches using the current workflow Items that represent an individual mass that is a mass entered by the user or a single peak mass from the spectrum have only one associated mass the
243. n the Results Browser Exclude Masses Workpad The Exclude Masses Workpad is a separate window that displays a list of items to exclude from any resubmitted searches using the current workflow Note Masses selected for exclusion are usually theoretical masses which can differ from masses found in the data Therefore due to the possibility of misassignment a detected mass being mistaken for a different theoretical mass the corresponding data is suppressed according to how well the masses match the theoretical masses rather than being completely extinguished To open the Exclude Masses Workpad right click either table and then click Exclude Re submit gt Open Exclude Mass Pad Exclude Masses Workpad Exclude Masses Workpad xi LDHB_ANAPL HNNVIPNGHFK RIDH_KLEAE YD41_HAEIN YFAX_ECOLI For other options in the Exclude Masses Workpad right click the workpad to display the menu Results browser 6 31 The menu items are lt Add Exclude There are four ways to add items to the Excluded Masses Workpad see Adding items to the excluded list on page 6 32 Delete Exclude Delete item from the Excluded list see Deleting items from the excluded list on page 6 33 Use Reagent Add an item that represents a digested protein or EST to the Excluded list see Running a simulated digest for a protein on page 6 33 View Exclude Masses View the mass values associated with an item see Viewing th
244. nal color when this dialog box was opened the bottom half shows the color currently selected The Reset button resets the color to the original 3 To set the color you have selected click OK Printing tab Use the Printing tab to view and edit the printing preferences Preferences dialog box Printing tab Preferences Renderer minimises white space Show content nodes in navigator tree Enable quick table pages Grid Size Default renderer ProteinLynx Renderer Restriction The dimmed options are not available in this version of PLGS 2 16 Setting up ProteinLynx Global SERVER To edit the printing preferences 1 To be able to add tabular as well as graphical data to a print template select the Enable quick table pages option This enables the option Tabular Data in the Template Type dialog box when creating new templates see Creating print templates on page 11 13 Selecting this also enables you to add tables to Results nodes table pages in the Print Tool navigator tree when creating new templates see Adding content to the results nodes on page 11 15 To change the size of the grid in the page editor view type or scroll to a number in the Grid Size option See Customizing print templates on page 11 19 for details of how to use the grid To change the print renderer for different applications select from the drop down list This changes the renderer for any new template
245. nd the data to produce a printed report a preview or an exported file Files can be exported as two types Comma separated values files csv HTML files html There are default templates supplied with PLGS or you can create your own using the Print Tool The Print Tool enables you to create modify and preview two types of template e Project template Prints details of all the hits in the project that have a score of higher than zero Workflow template Prints all details of the workflow used to obtain the data and a sorted list of proteins peptides and possibly masses Recommendation New users should use the default templates supplied If you are creating a template open a default template and save it as a new template Then edit the text graphics and so on The template editor enables you to edit and create print templates using an WYSIWYG What You See Is What You Get interface You use a properties editor to edit objects paragraphs images and so on Results pages are organized into hierarchical trees where you can apply limiting and sorting and then preview with the standard results set or any of your project data There are print wizards to print the data The print wizards are accessed from the navigator trees toolbar or results windows within the PLGS tools You can print project data from the navigator tree of any tool that shows the project name However you can only print workflow results fro
246. nd then click Set Sample see Setting a sample on page 5 11 7 Click OK The well changes color Setting processing parameters To set the processing parameters 1 Click Data Preparation 2 Click File gt New 3 Select Electrospray DDA and then click O 4 Give the Processing Parameters the title Data prep lt current date gt Each attribute set Mass Accuracy Noise Reduction Deisotoping and Centroiding has two attribute panels Electrospray Survey and MSMS A 14 Quick Start Tutorials In the Mass Accuracy Electrospray Survey panel set the attribute Perform Lock Spray Calibration to Yes Rule The Lock Spray Lock Mass of 785 8426 Da e the doubly charged ion of GFP is default in the software Mass Accuracy attributes Electrospray Survey lock spray Data Preparation Electrospray Survey Attribute Perform Lock Spray Calibration Yes Lock Spray Lock Mass 785 8426 Lock Spray Scans j2 Lock Mass tolerance 0 1 Da Perform Lock Spray Calibration Enable or disable Lock Spray calibration Enable for data acquired using an external Lock Spray interface Perform Lock Spray Calibration v In the Mass Accuracy MSMS panel set the attribute Perform Lock Spray Calibration to Yes Tip The Lock Spray Lock Mass of 716 4585 Da e the single charged ion of erythromycin is the default Mass Accuracy attributes MSMS lock spray Data Preparation Perform Lock
247. nd then click on one of these options Click Process Raw Data to submit the selected raw data for processing and then run the most recently attached workflow template Click Process Mass Spectrum to run the most recently attached workflow template for the selected processed data Changing Templates The processing parameters template associated with data can be changed in the sample list table and workflow templates added 5 7 To change processing parameters or add workflow templates 1 Click the row representing the data you wish to change or add a template to To select multiple rows hold Shift or Ctrl while clicking 2 Double click a cell in the Processing Parameters Template or Workflow Template column depending on which template setting you want to modify 3 Click the template you wish to associate with the selected data from the drop down list Tip If the template you want to use is not displayed in the list click the last item Choose new Processing Parameters Workflow Template from file then browse to the desired template Result All the selected rows are updated with the new selection 5 8 Specifying samples vials and plates with Container Manager Creating a new vial microtitre or target plate The following section describes the creation of a target plate The process for creating a new vial or microtitre plate is similar To create a new target plate 1 2 D ey In the navig
248. ng searching For details on how and when the assignments are made automatically see Automatic data curation on page B 7 Tip Modifying the assignment for a peptide will affect the assignments of its associated proteins or ESTs Peptide table OK miz z Peak mW Peptide m Delta Da Delta ppm Probability Start End Sequence 0 39 950 5081 1 949 5003 949 5021 ED 12747412 1 1273 7334 1273 7183 0 0151 1 30 39 R LLVVYPWTOR F PLG 2090 0291 1 2089 0212 2088 9463 0 0750 35 9 0 33 40 8 R FFESFGDLSTADAVNNPK V PLG Results browser 6 13 When the table is initially displayed or if the Workflow Results icon in the navigator tree is selected the table shows all of the peptides from each hit in the results When a hit is selected the table shows all of the peptides that belong to the selected hit When a protein or EST is selected the table shows all of the peptides that belong to the selected protein or EST The following operations can be performed using this table The columns to be displayed the order of columns and the precision with which numbers are shown can be controlled Individual peptides can be selected in the table or dragged and dropped into another component Controlling the columns in the tables 6 14 To add or remove columns in the tables 1 Right click the table 2 Click Select Table Columns 3 To add or remove a single column select or clear the check box for the
249. ns peptide or protein table 6 14 removing bookmarks 2 12 databanks 13 13 processors 2 9 search engines 2 8 Renew Period 13 8 13 10 replacing Import PlugIns 2 24 replicate filter 10 14 replicates viewing for a cluster protein 10 12 Report Monoisotopic Fragments attribute 8 15 required columns Ms sample list 16 7 requirements for sample lists 5 4 restoring archived databanks 13 15 backed up folder 1 5 1 11 old databanks 1 23 resubmitting search 6 15 results browser 6 3 export Expression 10 12 filter Expression 10 13 print Expression 10 13 viewing 6 2 results panel BLAST 14 27 retrieving databank entries 6 30 reviving databank archives 13 15 root folder 1 5 1 11 rtdb_monitor 15 15 running a simulated digest 6 29 MS sample list acquisition 16 8 on AIX 1 19 on the server 1 19 S Sample Editor 4 3 sample lists 5 2 A 11 columns 5 4 custom values 5 5 exporting 5 29 importing 5 3 processing and searching data 5 7 required columns M9 acquisition 16 7 requirements 5 4 view column 5 7 viewing 5 5 Sample Manager tool 4 2 5 11 description 4 2 samples adding 4 2 deleting 4 2 modifying 4 3 Index 15 organizing and annotating 5 11 viewing annotation 9 10 viewing information 5 23 Save button 5 22 7 4 8 3 12 5 12 10 13 12 saving digest reagents 12 10 modifier reagents 12 5 Savitzky Golay 8 10 Scans to Combine attribute 8 10 scoring MALDI B 4 matrices 14 25 matrix 14 25 schemes B 1 summary B 2 Sear
250. ntroiding attributes Continued Attribute Applies to Description TOF Resolution Electrospray Survey TOF resolution is m z divided MSMS by full peak width at half PSD MX maximum Used together with Electrospray MS the NP multiplier to correct for Low Energy detector deadtime High Energy NP Multiplier Electrospray Survey This attribute is used together MSMS PSD MX Electrospray MS Low Energy High Energy with TOF Resolution to correct for detector deadtime Minimum Charges to Report Low Energy The minimum charge state to report Contributions to ions from charge states lower than this value will be removed Recommendation A setting of 2 is recommended to reject singly charged noise Maximum Number of Charges Low Energy High Energy The maximum charge state to use in deisotoping This should be set to the maximum charge state that is commonly observed in the data to allow deisotoping to be performed correctly but no higher Increasing this value increases processing time 8 14 Creating custom processing parameters Peak Matching attributes The Peak Matching attributes are only available for PSD MX panels Peak Matching attributes Attribute Description Number of The number of ions to submit for peak matching The Precursors most intense ions in the spectrum are selected Fragment The intensity number of counts above which fragment Intensity pea
251. nts should be handled C 5 XML communication with the plugin implementation In order to allow easy integration with third party systems communication between the data storage system and the ProteinLynx system an XML based query language is defined in the ProteinLynx Document Type Definition DTD PLGS can communicate with a plugin by way of a series of predefined XML queries There are a series of query types Select nsert Delete e Update Within the DTD a set of elements related to querying XML and other documents is specified These elements constitute a primitive query language Essentially the Project Workflow and Mass Spectrum XML documents described in the DTD along with gel images sample lists and Expression Analysis experiments are the blocks of data by which the ProteinLynx system communicates For examples of the types of plugin specific queries see Basic plugin Specific Queries on page C 16 C 6 Implementing a plugin for ProteinLynx Global SERVER Adding a plugin to the PLGS application Once a new plugin has been created it needs to be added to the list of plugins in PLGS To add a plugin 1 Start the browser 2 Click Options gt Automation Setup 3 Click the PlugIns tab Automation Setup dialog box Plugins tab Pe Automation Setup lt co micromass plugin FileSystem FileSystemPluglin JavaPlugin uk co micromass plugin FileSystem 4 Click Add The PlugIn Selector dial
252. number of processors used can be individually set for three different circumstances DDA data processing Expression data processing Databank searching Caution Never set the number of processors to a value greater than the number of processors on your system DDA data processing Recommendation Make a copy of the file before editing as making changes other than those explicitly outlined below could prevent PLGS from operating properly To set the number of processors for DDA processing 1 Navigate to the 1ib directory underneath the PLGS installation directory 2 Open the process cfg file If it does not exist create a text file called process cfg and then open it 3 Add the following lines to the file MULTITASKING Number of Processors lt number gt Where lt number gt is the number of processors you want DDA processing to utilize 4 Save the file 1 24 Installing ProteinLynx Global SERVER Expression data processing Recommendation Make a copy of the file before editing as making changes other than those explicitly outlined below could prevent PLGS from operating properly To set the number of processors for Expression processing 1 Navigate to the 1ib directory underneath the PLGS installation directory 2 Open the process cfg file If it does not exist create a text file called process cfg and then open it 3 Add the following lines to the file EKL Processing Numb
253. o those identified for use in Expression experiments To open the Sample Manager click the Sample Manager icon on the tool tray Adding a sample To add a sample to a project 1 In the navigator tree click and then right click Original Samples 2 Click Add New Sample 3 You are asked whether you want to add the new sample to a new vial Click Yes or No Rationale Whether you choose Yes or No a new sample is produced its details are displayed and it is added to the navigator tree Clicking Yes also produces a new vial in the Container Manager to which the new sample is added Deleting a sample 4 2 To delete a sample 1 Click a sample in the navigator tree 2 Click Delete on the toolbar Restriction You can only delete samples that are not being used anywhere else on the system Annotating and tracking samples with Sample Manager Sample editor To modify or view the information associated with a sample highlight the sample name in the navigator tree The Sample Editor is displayed Sample Manager sample editor Sample Manager Sample 1 Select Attribute Tue Jan 28 11 34 43 GMT 0 Databank Hyperlinks Enter the name of the sample Name Sample 1 Enter Value To add or modify an attribute 1 Click the attribute in the panel 2 Enter the value at the bottom of the panel Restriction You cannot modify the Date attribute 4 3 The foll
254. odifications Yellow A peptide that contains post translational modifications and missed cleavage sites Fragment ion display for MSMS data a 60 045 169113 272197 359 229 430 267 569 309 672 393 769 446 883 489 996 573 1159 636 0 021 0 005 0 014 0 031 0 017 0 044 0 078 b 88 04 187 108 300 192 387 224 458 261 587 304 700 388 797 441 911 484 1024 568 1187 631 0 016 0 016 0 003 0 008 60 087 0 04 EE eS ee ee 38 45 38 43 85 73 39 9 99 9 99 87 99 87 84 12 46 2 43 56 77 61 99 86 y 1274 711 1175 642 1062 558 975 526 904 489 775 447 662 363 565 31 451 267 338 183 175 11 0 062 0 012 0 01 60 029 0 02 0 029 0 003 0 001 z 1257 684 1158 616 1045 532 958 5 887 463 75842 645 336 548 283 434 24 321 156 158 09 0 081 0 013 0 04 0 01 0 023 0 005 Spectrum Viewer for MSMS data For a search with an MSMS spectrum the bottom component of the results browser is a graphical display of the fragmentation spectrum for the current parent peak Results browser 6 21 Spectrum Viewer for MSMS data Precursor mass 471 81 charge 2 4 bMax ENI _J yMax a w 5 max You cannot directly select results in the Spectrum Viewer However the viewer responds to selections in the other browser components Ifa mass is selected the fragmentation spectrum for the corresponding peak is displayed Ifa peptide is selected th
255. og box opens in which you can set up either an Executable or Java Class type of plugin C 7 Plugin Selector dialog box Executable plugin type Plugin Selector x C 8 Implementing a plugin for ProteinLynx Global SERVER Plugin Selector dialog box Java Class plugin type Plugin Selector Add Modify Remove _ Enable Export Selected Results from Container v Enable Save Projects via this Plugin or come 5 Select either an Executable or Java Class type of plugin and set the parameters 6 Once added successfully the new plugin is displayed in the Exports list C 9 Plugins page Plugin displayed in Exports list Ja Automation Setup lt co micromass plugin FileSystem FileSystemPlugin JavaPlugin uk co micromass plugin FileSystem When an item is saved it will be passed to the new plugin as well as to the default FileSystemPlugIn C 10 Implementing a plugin for ProteinLynx Global SERVER An example Executable plugin The following is the source required to create an example Executable plugin called HelloPlugIn exe Build this code in Visual Studio to create the executable and then add it to the exports in PLGS The HelloPlugIn exe takes the input to the plugin and then prints it out to a file called helloplugin1 txt which can be found in the working directory you set when adding the plugin to the list of export plugins Try this and see how it works HelloPlugin1 Reads
256. ommon Desktop Environment a graphical user interface In a terminal window the prompt symbol indicates what shell you are using The and amp respectively represent the Korn Bourne and C shells Check if the TMPDIR variable exists Setting the TMPDIR creates a pointer to a location where there is sufficient space for working files At the prompt type the command env pg Press Enter The environmental variables are displayed UNIX Help for Installing PLGS on AIX Platforms Example TMPDIR usr tmp myid dot LANG En_US UNAME davisd PAGER bin pg VISUAL vi PATH usr ucb usr t u dot bin u binl MAILPATH usr mail dot MAT LRECORD u dot Ou EXINIT set beautify noflash nomesg report 1 showmode showmatch EDITOR vi PSCH gt HISTFIL LOGNAME dot MAIL usr mail dot E u dot history PSl dot davisd PWD gt PS3 PS2 gt epath USER dot usr bin SHELL bin ksh HISTSIZ F 500 HOME u dot FCEDIT vi TERM 1f MATLMSG YOU HAVE YOUR PW NEW MA D u dot ENV u dot env L USE t dot has mail tmail TH E mail COMMAND TO S pp X11 bin bin usr bin etc u do zal i D 3 Adding TMPDIR To add TMPDIR 1 Type the commands TMPDIR Where ever you have large space allocation on system ex
257. on UNIX Before installing PLGS on UNIX you must complete these tasks Back up the PLGS directories Ensure that you are logged on with root permissions Uninstall previous versions of PLGS Backing up the PLGS directory Before installing PLGS make a backup copy of the PLGS directory In a terminal window type cp R lt source folder gt lt destination folder gt Uninstalling a previous version of PLGS To uninstall a previous version of PLGS using the command prompt 1 Go to the old version s _uninst directory by typing cd _uninst 2 Run the uninstaller by typing uninstall bin 3 Follow the instructions in the Uninstall wizard Tip After uninstalling PLGS errors can be reported This is usually due to the uninstaller not being able to remove the uninstaller resources This is caused by the user running the uninstaller binary from within the _uninst directory This means that you will have to remove the _uninst and main PLGS directories manually Installing PLGS on UNIX To install PLGS on UNIX 1 Insert the PLGS installer CD into the drive Recommendation Before initializing the installer copy the installer package from the CD to the local file system Mount the CD using SMIT or manually using the mount command See also For instructions for mounting the CD see Appendix D UNIX Help for Installing PLGS on AIX Platforms Type the following command in the installer directory cp PLGS2 2 5 lt unix
258. on the PLmicrokernel file are sufficient If not change the permissions by typing the following command in the file chmod 777 PLmicrokernel Check that the number of processors specified in the config micro mkconfig file are appropriate see Setting the number of processors on page 1 24 Ensure you are logged on as root Ensure user root has read write and execute permissions on the databanks and their associated files Recommendation Index files that are created by databanks should be in the same directory as the databanks Search engine failures If error traces are seen in the console window or log file of the search engine ensure that you have selected the correct format for all databanks added to the server see Databank attributes on page 13 4 Large databank gt 2 GB problems If you experience problems when searching or adding large databanks check the following Check that large file support is enabled on the temporary space the directory is specified in the search engine startup script Check that large file support is enabled on the directory that contains the databanks Check that the search engine has 2 GB of RAM allocated to it See Search engine memory allocation on page 1 17 for details Databank and BLAST searching problems If problems occur with databank or BLAST searching try carrying out the following operations Remove user account file size restrictions 1 21 Increase the amount o
259. on to other specified filters 11 16 Creating print templates and printing project data Example If you apply two combined filters to the results the report only shows a condition for example a protein that satisfies both filters if the same two filters are applied as additional the protein is shown if it satisfies either filter Sorting results To add sorting fields click the Sorting tab and then click Add Properties dialog box specifying a sort Filtering Limiting Container Name Add Position LS Ji Select Sort Field x Ascending JUL Container Name Descending Sample Name Click fields in the list and then select to sort in either ascending or descending order Limiting results To enable limiting click the Limiting tab and then select the Enable Limiting check box 11 17 Properties dialog box Limiting tab Filtering Sorting Enable Limiting Number of results Use this tab to limit the number of results that are returned for proteins peptides and so on 11 18 Creating print templates and printing project data Customizing print templates You can add pages that contain text fields and graphics elements images and horizontal rules to customize the style of the report For example you can add a company logo standard company information page numbers and so on In the following examples you will create a new page f
260. only recently added sequences Performing updates reduces the need for frequent full downloads of databanks which can use a lot of resources If this attribute is set to true you must set the Update URL Address attribute to specify a remote location URL from which the databank will be periodically updated There are several other options relating to periodic updating that can be set or be left at their default values Update Compression Type e Update Renew Period Keep Archives Processing Start Time e Processing End Time Update URL Address This field must be set if the Periodically Update attribute has been set to True This field contains the URL address from which the databank should be periodically updated 1 Click the URL button to open the URL Chooser dialog box 2 Type the URL address of the remote file from which the databank should be periodically updated and then click Open The system locates the remote address and checks that it can be accessed This can take a few seconds Update Compression Type Rule This option is only available if the Periodically Update attribute has been set to True The details of this attribute are the same as for Download Compression Type 13 9 Databank attributes Continued Attribute Update Renew Period Description Rule This option is only available if the Periodically Update attribute has been set to True Enter the number of d
261. ools Now consider that only substitutions are being considered no modifications and that all substitutions are valid Each residue can therefore undergo 19 different substitutions Considering a maximum of 0 mods subs per peptide will generate only 1 peptide the starting peptide above Setting max mods subs to 1 will generate 191 10 x 19 1 potential matching peptides Considering a maximum of 2 mods subs per peptide will now generate 16436 45 x 19 x 19 10 x 19 1 potential matching peptides Therefore the number of potential peptides grows rapidly making AutoMod a powerful tool in matching peptides that are missed by conventional databank searching To ensure that the tool is used efficiently you must take care to limit this value to a sensible figure and to assign the peptide tolerance appropriately Default By default each peptide is allowed to contain one modification or substitution Specifying the likelihood of substitutions The likelihood of each individual amino acid substitution has been calculated in the generation of the Blosum62 matrix and is represented as a score from 4 to 11 4 being an unlikely substitution and 11 being the most likely For example substitution of a methionine for a leucine has a score of 2 substitution of a tryptophan for a proline has a score of 4 In the text box type a value between 4 and 11 This limits the number of substitutions considered to those that have a hig
262. oping and Centroiding 8 5 Mass Accuracy 8 5 Noise Reduction 8 5 Peak Matching 8 5 creating 8 2 description 8 2 methods to acquire data 8 5 removing 8 4 saving 8 3 Processing Start Time databank attribute 13 10 processors host 2 20 modifying 2 9 port 2 20 removing 2 9 Progenesis XML file 3 3 Index 14 importing gels from 9 6 program group 1 5 project template printing 11 2 projects 3 1 closing 3 6 creating 3 2 5 3 10 2 deleting 3 6 exporting 3 3 importing 3 3 opening 3 5 updating 3 5 Protein Expression 10 2 protein mass 14 8 protein sequences selecting for search 14 18 Protein table 6 12 10 9 10 13 add remove columns 6 14 change column order 6 15 view replicates 10 12 viewing 10 10 Protein view 6 4 6 7 Protein Workpad 6 27 digest fragments 6 30 ProteinLynx Browser Automation Setup dialog box 2 18 ProteinLynx Browser Preferences dialog box 2 5 5 33 Q quantitation assess data quality 10 25 quantitation analysis Expression experiment 10 8 Quantitation Reagent attribute 12 5 query tools description 14 1 toolbars 14 2 question mark 6 5 OK column 6 12 R Range Units attribute 8 16 ratio filter 10 15 raw data 5 17 attaching 5 13 reagents modifier 14 21 Real Time data processing 15 14 databank searching 15 1 15 8 setting up 15 8 displaying diagnostics 15 15 menu 15 8 status 15 7 15 9 real time status 15 10 remote searching 15 14 Remove button 2 8 2 9 2 12 7 4 8 4 13 13 remove add colum
263. or an introduction and add text graphics and fields to the header footer and the introduction page The examples illustrate the kind of objects that can be added to a template you can insert a paragraph field image or horizontal rule anywhere on any page Prerequisite The following sections assume that a print template is open for modification You can customize one of the built in templates or work with one you created yourself see Creating print templates on page 11 18 To add pages 1 Inthe template navigator tree right click the Introduction node and then click Insert gt Page 2 Inthe tree right click the new page and then click Rename Change the page name to Template Details When adding pages you can display a grid which helps you to locate and align the elements To display the grid 1 Inthe menu bar click View gt Toggle Grid 2 Change the size of the grid in the Preferences dialog box see Printing tab on page 2 16 To add paragraphs 1 Inthe template navigator tree right click the Header node 2 Click Insert gt Paragraph Tip This method inserts the paragraph in a default location and with a default size in the page To insert a paragraph box in a location and with a size of your choice use the buttons on the right of the browser screen 11 19 For more details on using the buttons see Buttons for adding content to pages on page 11 23 Print Tool adding paragraphs YA Prot
264. or manually See Chapter 1 Installing ProteinLynx Global SERVER for details Run the microkernel search engine from the command by typing PLmicrokernel exe MassLynxURL Remote URL Example If the MassLynx PC has the URL 10 1 14 85 and the URL of the PC on which you are running the search engine is 10 1 11 198 type PLmicrokernel exe 10 1 14 85 10 1 11 198 Requirement You must know the URL of both this PC and the MassLynx acquisition PC When the program enters the wait state it is ready to take input from the MassLynx PC Displaying diagnostics Diagnostic windows display processing and search information It is not usually necessary to have these windows visible To display the diagnostic windows click Help gt Show Diagnostics Caution Do not close the diagnostic windows by clicking the close buttons at the top right corner as doing so can cause the applications to terminate Instead click Help gt Hide Diagnostics If you have a local microkernel search engine three diagnostic windows are displayed PLmicrokernel search window for displaying the state of the database search engine process_kernel window for displaying the state of the raw data processing module rtdb_monitor window for displaying the state of the module responsible for monitoring processed spectra and submitting these spectra to the microkernel 15 15 Rule These windows will only be displayed if you have enabled the search en
265. oteinLynx DTD sssessesssosssossososoosssosscoosoo C 21 Plugin Process exit GOMES siisiicisesciiinicadencsesiectcnacadssanndnadcinrdsiacseadicdiacaeuaceds C 26 UML Class Diagram for the PLGS plugin Architecture 000 C 27 D UNIX Help for Installing PLGS on AIX Platforms D 1 Installing PLGS using the command line sesssessssssessesoseossecoecooecosesoesoeseose D 2 Addas TAEDA D 4 Mounino aC ROW a tvecoawasaectineuniaaansretaiadevetoanaen D 4 Uone oE Meee e eer peeennn ir re tu cnarnlr a ty arent ay re Per D 6 Using navigation and installation COMMANAG cc0cseeeseeessesseesseersesteeees D 8 Creating and managing user accounts and QYOUPG ssccccecceessssstteeeeeeeees D 9 Table of Contents xvii E Databanks Formats sssssssesssssscssccososssesssosscosecossossossscsseosssossossosssesse E 1 URLaddress s cernas aeaa r aa e E E E EEES EENES E 2 SPTREMBL flat file format dcssssscascessesed cussccescecanssaseassdesstecesdesauseaseesesesseesansaseess E 3 Genbank flat file format s0cissssdesdedsescdeccestessssessuensaee a ae sS EEE NE O Er A aE E 6 BLAST flat file format esnearen e e o iia E 8 FASTA flat file format siscsssncvessixpincssassssscsncaatessnceewetenisiscnsencmeasssnxnatnacnanissaneesesens E 9 FASTA STANDARD ceria concdesentesenessianalsenictacwteaadee sae EAR E IET E R SRS E 9 FASTA NCBI_EXPASY_STANDARD cccccsssssssscecccesssesseeceecccesesseseeeees E
266. ow Results uh 900 777 1 899 769 uh 902 769 1 901 761 ut 904 808 1 903 8 uh 916 761 1 915 753 uh 1049 849 1 1048 841 uh 1065 804 1 1064 796 ut 1071 828 1 1070 82 iit 1081 77 1 1080 762 wie n e LLLQNLVIR n AIAQVIPELK uh 1087 788 1 1086 78 uh 1089 779 1 1088 771 ob asaan anam a ann ara Selecting items in the navigator tree To select any item in the navigator tree click the node that represents the item The other components in the results browser update automatically to reflect the selection Selecting one item can cause other items to be selected Results browser 6 9 Example If a peptide is selected the hit protein and peak mass to which the peptide is matched are also selected Results of selecting navigator tree nodes Icon Selected Result Workflow All selections are reset The protein table shows the results top scoring protein or EST from each hit The peptide table shows the peptides matched to all of the top scoring proteins and ESTs The MS spectrum display will color the peaks matched to peptides from the top scoring protein or EST in the results The MS MS spectrum display will show fragmentation data for the first peptide in the peptide table Protein or EST The protein table shows all proteins and ESTs that belong to the same hit as the selection and the row showing the selected protein or EST is highlighted The peptide table shows all peptides tha
267. ow template files processing parameters files and so on root contains project files that you have created Backing up databanks If any of your databanks are stored in the directory in which PLGS is installed you must make backups of the databanks before uninstalling PLGS Uninstalling PLGS in Windows To uninstall a previous version of PLGS 1 From the ProteinLynx program group select the uninstall option ProteinLynx program group uninstall option fin ProteinLynx Ynn amp ProteinLynx Browser ProteinLynx Microkernel G ProteinLynx Processor Engine ET ProteinLynx Search Engine gj Uninstall PLGS n n 1 3 Exception If you are uninstalling PLGS 2 2 5 the Microkernel Processor Engine and Search Engine options are not displayed in the program group Follow the instructions in the Uninstaller wizard Installing PLGS on Windows To install PLGS on Windows 1 Double click the PLGS2 2 5_WINDOWS exe file to open the InstallShield Wizard Result After a short pause the ProteinLynx Global SERVER installation wizard will be displayed Click Next Read and understand the terms of the license agreement select the accept option and then click Next In the product destination screen do one of the these actions Click Next to accept the default installation location C PLGS lt version number gt Browse for another directory and then click Next If the installer cannot detect a val
268. owing table details the attribute settings Sample Manager sample editor parameters with drop down lists Attributes Description Sex This can be set to UNKNOWN MALE or FEMALE Condition This can be set to UNKNOWN NORMAL CHALLENGED PERTURBED MODIFIED and AFFECTED Tag This is the isotope label used in an Expression Analysis experiment For samples that are not involved in quantification studies this value will not be set While this value can be set using this tool it is more appropriate to set it in the Expression Analysis tool Databank To attach a databank hyperlink to a sample Hyperlinks 1 Click the Databank field and then click a database in the list 2 In the Unique Identifier field enter the unique identifier of the required databank entry 3 Click the Save button to add the hyperlink Alternative Click the New button to save the current hyperlink and create a new row in which another hyperlink can be entered Requirement For a databank to appear in the list its URL must be entered as a bookmark see Bookmarks tab on page 2 11 and set as non static Using SWISS PROT TrEMBL as an example it is necessary to enter an accession number in the Unique Identifier field to generate a valid hyperlink 4 4 Annotating and tracking samples with Sample Manager Generating processed samples Any number of samples can be mixed together to produce a processed sample Selected samples a
269. owser 14 19 De Novo Sequencing parameters Databank Search DeNovo Sequencing Parameters Mass Spectrum None Fragment Tolerance Estimated Calibration Error Maximum Hits to Return 4utoMod Analysis 0 005 Da Primary Digest Reagent Trypsin Secondary Digest Reagent None De Novo Sequencing Modifications validate Results Q Mass Spectrum BLAST Searching Choose the MassSpectrum XML file Mass Spectrum ee Data Preparation File URL Clear ENS Workflow Designer E Q Roane nO De Novo Sequencing To perform De Novo sequencing 1 Click an attribute in the table see De Novo sequencing parameters on page 14 21 for details and then edit the value in the panel at the bottom of the table 2 When the required fields have been edited click the Submit button gt on the toolbar to start the search When the analysis is complete the results are displayed in the unified results panel that is added to the desktop 14 20 Query Tools De Novo sequencing parameters The following sections detail the attributes in the De Novo Sequencing Parameters table The parameters Mass Spectrum Fragment Tolerance Primary Digest Reagent Secondary Digest Reagent are described in Databank search parameters on page 14 5 Specifying the estimated calibration error This value is fundamental to the scoring of a peptide sequence against a given fragmentation s
270. ox in the toolbar and then clicking the name of the project 3 Click Edit gt Run SuperTrack Result The SuperTrack Manager is displayed 5 26 Specifying samples vials and plates with Container Manager SuperTrack Manager mm SuperTrack Manager 2 2 Fine Delta RT Minutes 0 05 Coarse Delta RT Minutes 2 0 Sample Select Replicate Container Position CSF260805_011 4 A2 E _CSF260805_012 4 A3 CSF260805_010 4 A1 407 Default KIKIR The SuperTrack Manager provides access to several settings Fine Delta retention time the retention time tolerance for a replicate reflecting the precision with which retention time can be estimated within a single function such as high energy Coarse Delta retention time the retention time tolerance between replicates reflecting the reproducibility of retention time across different injections of the same sample Project samples as defined in Sample Manager see Annotating and tracking samples with Sample Manager on page 4 1 Replicates associated with the selected samples To run SuperTrack 1 Select check boxes beside the project samples of interest 2 Select the check boxes beside the replicates you want to SuperTrack 3 Click Go Result SuperTrack spectrum nodes appear in the Container Manager tree for each selected sample Processing can take some time progress is shown at the bottom righ
271. page 10 3 for details 2 Inthe Data View column click either the bar chart or scatter chart icon To set the values for axes 1 To set the values displayed on the y axis click 2 To set the values for the x axis click A 2 Click the values that you want to display on that axis To alter the range displayed on an axis 1 To modify the lower limit of the range click and hold the left or bottom axis slider To modify the upper limit of the range click and hold the right or top axis slider Axis slider Axis slider Al 2 Drag the slider to modify the range limit To switch between bar chart and scatter chart view Click luu to show the bar chart view Click A to show the scatter chart view 10 25 To show hide the EMRT and Peptide panes Click il to show hide the EMRT Clusters pane Click E to show hide the Matching Peptides pane 10 26 Using Expression Analysis to compare and analyze sample groups 11 Creating print templates and printing project data The Print Tool enables the creation and modification of printing templates Printing templates are used to control how project data is printed Contents Topic Page Printing data 11 2 Using print wizards 11 3 Opening and deleting print templates 11 12 Creating print templates 11 13 Customizing print templates 11 19 Printing data When you print data you combine project or workflow data with a template Rendering combines the template a
272. parameters node directly below the target plate name is displayed see Adding processing parameters templates on page 5 21 A 18 Quick Start Tutorials 2 3 Click and then right click the Default node Click Change Processing Parameters Processing Parameters Templates dialog box Processing Parameters Templates Select Processing Parameters Template Choose new Processing Parameters Template from file Y Default MALDI MS Click Choose new processing parameters template from file and then click OK Click the processing parameter file Data prep lt date gt xml that you created earlier see Setting processing parameters on page A 14 and then click Open Attaching the workflow file To attach the workflow file 1 In Container Manager highlight all the wells on the plate for which you have set samples see Setting the target plate on page A 5 by dragging a rectangle over them Right click Select Set Attached Templates gt Workflow Template to Mass Spectrum Click OK to Choose a new Workflow Template from file Click the Q Tof workflow file Workflow lt date gt xml that you created earlier see Creating a workflow on page A 17 and then click Open Exporting the sample list to MassLynx For further details see Exporting a sample list to MassLynx on page 5 29 To export the sample list 1 In Container Manager right click on the target plate node and then click Export Sa
273. pectrum A tight error will significantly reward well measured data in the scoring so it is reeommended that spectra submitted are well mass measured to allow a low estimated calibration error to be set It is not necessary to adjust the estimated calibration error for small variations of this number in the fourth decimal place This value will be combined with the estimated mass measurement error for each peak The estimated mass measurement error is calculated by the processor To specify an estimated calibration error type the value into the text field and then select the units from the combo box Available units are Daltons Da and parts per million ppm Specifying maximum hits to return The Maximum Hits to Return attribute corresponds to the maximum number of De Novo sequenced peptides to return per fragmentation spectrum If the Validate Results feature is used only those peptides that are validated will be returned It is therefore possible that fewer sequences are returned for some spectra than the value specified here Specifying modifications to peptides Specifying modifications is optional By default no modifications are applied to the peptides produced by the digest The Modifications list contains all the available modifier reagents 14 21 De Novo Sequencing parameters Modifications list Modifications Select the modifications to be considered from the list Modifications Acetyl K a Acetyl N TER
274. peptide or protein table 6 14 Change Processing Parameters command 5 21 changing preferences 2 5 processing parameters 5 7 Chromatogram attribute set 8 5 8 15 circled gel spots 9 9 Clear OK assignments 6 5 client installation 1 3 starting PLGS 1 5 client server environment installation 1 1 closing projects 3 6 clusters import significant 10 24 include or exclude 10 13 Coarse Delta retention time 5 27 columns displaying 6 14 Combine Options attribute 8 10 commands Index 3 Change Processing Parameters 5 21 Import Worksheet 5 30 A 10 A 20 Microkernel Search Engine 15 15 Process Raw Data 5 17 Compression Type 13 8 13 9 confidence limit filter 10 15 connecting to search engine 13 17 Consider Modifications parameter 14 16 Consider Substitutions parameter 14 16 Container Manager 5 2 copying data 6 16 6 26 creating databanks 13 3 Expression experiment 10 3 new project 3 2 A 2 new target plate 5 9 project 5 3 10 2 target plate 5 9 workflows A 7 A 17 cross OK column 6 12 curated filter print templates 11 16 curation automatic 6 12 B 7 data 6 5 of data 10 11 D data acquisition 15 1 A 11 A 21 curation 10 11 automatic 6 12 B 7 Expression 16 1 file 14 5 Index 4 graphical 11 14 MS 16 1 printing 11 2 processing 15 14 tabular 11 14 data directed analysis DDA A 23 chromatograms A 22 Data Preparation tool attribute sets 8 5 creating a new processing parameters template 8 2 definition o
275. periment Attributes This section names the Expression analysis and specifies a description of its purpose 10 4 Using Expression Analysis to compare and analyze sample groups Select Grouping Method Use this section to specify how samples should be grouped Groups are compared against one another Choose the processed sample see Generating processed samples on page 4 5 that contains the samples you wish to use in the experiment If the optional Waters Protein Expression System is used you can clear the Use isotope labelled sample box and choose any sample not just processed samples Rules for isotope labeled experiments Only samples that have been labeled in Sample Manager using the Tag field appear in the drop down list Grouping methods other than placing the samples into separate groups are available only when there are more than two samples in the processed sample selected Grouping methods Method Place samples into separate groups How to 1 Click Place samples in separate groups 2 In the list click the samples you want to include in the analysis Click Select All to include all the samples or use Ctrl or Shift to select multiple samples 3 Click Apply Result Each sample is in its own group 10 5 Grouping methods Method How to Result Group by 1 Click Group by experiment Samples that share experiment variable the selected variabl
276. permissions on page 1 8 Uninstall previous versions of PLGS see Uninstalling previous versions of PLGS in Linux on page 1 8 Backing up the PLGS folders Before installing PLGS make a backup copy of the following folders e docs root Backing up databanks If any of your databanks are stored in the directory in which PLGS is installed you must make backups of the databanks before uninstalling PLGS 1 7 Changing file permissions File permissions exist on Linux to prevent unauthorized access Before installing PLGS ensure you are logged on with user ROOT permissions If file permissions problems continue you need to change the file permissions To change a file s permissions 1 Logon as the root user 2 Use the cd command to navigate to the file s folder 3 Change the file s permission settings by typing chmod 777 filename This removes the restrictions on all file permissions Uninstalling previous versions of PLGS in Linux Previous version of PLGS can be uninstalled from a command prompt or by using the GUI The uninstaller deletes all folders and contents that were installed with PLGS and any folders and files that you created using PLGS To uninstall PLGS using the command prompt 1 Open a terminal window and type cd PLGS INSTALL FOLDER _uninst This takes you to the uninstall folder 2 Torun the uninstaller program type uninstall bin 3 Follow the instructions in the Uninstaller wiz
277. ple if searching is being performed on a remote server do not block on results as the acquisition PC would be free to continue acquisition during the data search step Processor Host Type the IP address of the computer on which the processor is running The processor module handles processing of raw data to produce mass spectra Tip This information is for the local processor Use the Preferences dialog box see Processors tab on page 2 8 to specify remote processors Processor Port Type the port number used by the processor module Spectrum Output tab The Spectrum Output tab enables you to specify additional formats in which spectra can be saved after processing Spectra are automatically saved in ProteinLynx XML format 2 20 Setting up ProteinLynx Global SERVER Automation Setup dialog box Spectrum Output tab ICUME 1 COLLIN 1 LOCALS 1 Temp ICUME 1 COLLIN 1 LOCALS 1 Temp CUME 1 COLLIN 11LOCALS 1 Temp Ei si row on coco 2 21 You can set the following parameters Spectrum Output tab parameters Parameter DTA Output Description DTA format is a Waters file format for storing MS MS spectra The first line of a DTA format file contains the singly protonated peptide mass MH and the peptide charge state as a pair of space separated values Subsequent lines contain space separated pairs of fragment ion m z and intensity values In a DTA file the precursor peptid
278. port TMPDIR 2 Type env pg This verifies that the TMPDIR path has been set correctly Mounting a CD ROM To mount a CD ROM 1 Insert the CD and then at the command prompt type mount cdrom 2 Press Enter This mounts the CD ROM on the file system cdrom The CD ROM drive should spin up If you type the command incorrectly or omit the an error will occur D 4 UNIX Help for Installing PLGS on AIX Platforms O A x KK KKK KKK KKK KKK KKK KK KKK KEKE KE KKK KE 3 To verify you have mounted the CD type the commands cd cdrom pwd ls a The contents of the CD should be listed If the CD ROM does not mount go to SMIT to check what the CD ROM drive is referenced as Open SMIT Select System Storage Management Physical amp Logical Storage 1 2 3 Select File Systems 4 Select List All File Systems 5 In the list locate the device dev cd0 The mount point is the reference to be used 6 Click Done D 6 UNIX Help for Installing PLGS on AIX Platforms 7 Select List All Mounted File Systems The device dev cd0 should be mounted 8 Click Done If the CD ROM drive is not mounted you can mount it by selecting Mount A File System and then selecting dev cd0 from the list H X lt Entry Fi FILE SYSTEM name i dev calf r over which to mount 0 To remove the disk you will need to unmount the CD using SMIT or type unmount usr cdrom D
279. raw data 5 13 workflow file A 9 A 19 workflow templates 5 20 attribute sets Index 1 Chromatogram 8 5 8 15 Deisotoping and Centroiding 8 5 A 14 Mass Accuracy 8 5 A 14 Noise Reduction 8 5 A 14 Peak Matching 8 5 attributes Applies to 12 5 Automatic Thresholds 8 13 8 16 Background Polynomial 8 10 Background Subtract Type 8 9 Background Threshold 8 9 Calibration File 8 15 Centroid Top 8 13 Combine Options 8 10 databank 13 4 Deisotoping type 8 12 Delta Mass 12 5 Expected Peak Width 8 15 External Lock Mass 8 6 Fragment Intensity Threshold 8 15 Fragment Matching Window 8 15 Fragments 12 5 Intensity Range 8 11 Intensity Threshold 8 7 Iterations 8 12 Lock Mass tolerance 8 7 Lock Spray Lock Mass 8 8 Lock Spray Scans 8 8 Low Mass Threshold 8 11 Maximum Number of Charges 8 14 Minimum Charges to Report 8 14 Minimum Peak Width 8 13 8 15 Modifier type 12 5 Name 12 4 NP Multiplier 8 14 Number of Precursors 8 15 Peak Width Units 8 16 Peptide Filter 8 11 Index 2 Perform Deisotoping 8 12 Perform Lock Spray Calibration 8 8 Perform Smoothing 8 10 Precursor Matching Window 8 15 Primary Internal Lock Mass 8 7 Quantitation Reagent 12 5 Range Units 8 16 Report Monoisotopic Fragments 8 15 Scans to Combine 8 10 Secondary Internal Lock Mass 8 7 Select Calibration Type 8 6 Select start time 8 16 Select stop time 8 16 Select time range 8 16 Smoothing Iterations 8 10 Smoothing Type 8 10 Smoothing Window 8 10 Threshold 8 13 8
280. re automatically generated into processed samples Processed samples can be used in Expression Analysis To generate a processed sample 1 Select two or more original samples use Shift or Ctrl while selecting and then right click 2 Click Generate Processed Sample A new sample is produced and added below the Processed Samples node The samples from which the new processed sample is generated are also listed in the navigator tree You can annotate the new sample 4 5 4 6 Annotating and tracking samples with Sample Manager Specifying samples vials and plates with Container Manager Container Manager is fundamental to ProteinLynx Global SERVER It enables you to perform a number of operations Specify the samples and data you want to analyze Attach templates that determine how data is processed Start processing Access your results Understanding Container Manager is the quickest way to get up and running with PLGS Requirement Specify your instrument before beginning to use Container Manager see Instrument tab on page 2 10 Contents Topic Page What is Container Manager 5 2 Importing and viewing PLGS sample lists 5 3 Creating a new vial microtitre or target plate 5 9 Setting a sample 5 11 Attaching raw data 5 13 Processing raw data 5 17 Re searching processed data 5 20 Adding processing parameters templates 5 21 Exporting and importing mass spectra 5 22 Working with plates 5 23 Simplifying
281. ream char buf new char 1024 int nRead 0 StringBuffer buffer new StringBufferQ System out println Here comes the input to the MirrorPlugIn do nRead reader read buf 0 buf length if nRead gt 0 los write Buf 0 nRead buffer append buf 0 nRead while nRead 1 System out println buffer toString System out println The MirrorPlugIn has finished return 0 This method is used to set any properties the PlugIn may have public void setProperties Properties properties To do Auto generated method stub Basic plugin Specific Queries There are four basic plugin specific queries Selection of elements Update of elements Deletion of elements JInsertion of documents Selection of elements C 16 lt xml version 1 0 gt lt QUERY gt lt SELECT ELEMENT_TYPE PROJECT RETURN document gt lt REFERENCE NAME PROJECT gt lt REF_ATTRIBUTE NAME PROJECT_ID VALUE Project3 gt lt REFERENCE gt lt SELECT gt lt QUERY gt Selecting a Project document for a given Project ID Above is an example query to the FileSystemPlugIn The query is asking the plugin to select the Project with the Project ID of Project 3 This example clearly illustrates how simple queries can be built All queries have an outer lt QUERY gt tag and within this tag will be a series of descriptive elements to define the query In
282. rectory Type a description of the search engine in the Description text box To connect immediately select Connect If you want the search engine to keep running when the ProteinLynx browser is closed select Detach Click OK Modifying a search engine You can modify the type of search engine IP address description and the connection details of a search engine To modify a search engine 1 Double click the search engine in the list Alternative Click the search engine and then click Modify The Modify Search Engine dialog box opens which has the same fields as the Add Search Engine dialog box Modify the details as required Click OK 2 7 Removing a search engine To remove a search engine click the search engine and then click Remove Processors tab Use this tab to add modify or remove local or remote processors The browser can process raw data on the host machine or on remote processors However the Processor module must be running on the same computer as the raw data The details of any remote processor must be entered in the Processors page on the host machine Preferences dialog box Processors tab Preferences Add Modify ll Remove Adding a processor You can add local or remote processors 2 8 Setting up ProteinLynx Global SERVER To add a processor 1 2 6 Click Add In the Address text box type or paste the IP address of the computer on whic
283. results for that sequence are displayed in the lower section of the window To see the alignment for a hit scroll down in the lower section of the BLAST Results Panel Alternatively click on the hyperlink of one of the matches to jump to the alignment details for that hit 14 27 14 28 Query Tools 1 5 Real Time Databank Searching The Real Time Databank searching application allows the acquisition system or more particularly a data dependent acquisition DDA to be updated according to the results obtained from a databank search Specifically if a protein is identified while a data dependent acquisition is in progress the software generates all the peptide masses corresponding to the identified protein The acquisition system then uses these masses to form an exclude list to prevent any further MSMS data collection for that particular protein Real Time Databank searching is accessed from within MassLynx See also Some familiarity with MassLynx is recommended Refer to the MassLynx Getting Started Guide and the MassLynx Help for information on using the MassLynx window sample lists and the MassLynx queue You will also need to refer to the Data Acquisition sections of the MassLynx Help or relevant Operator s Guide Rule Real Time Databank searching is only available for MassLynx versions 4 0 SP1 and later Contents Topic Page Using real time databank searching 15 2 Advanced options 15 14 15 1 Using re
284. rkflow queue 26 02 03 11 14 12 STATUS There are no jobs in the processor queue 26 02 03 11 14 12 STATUS Waiting for next processing job 26 02 03 11 14 12 INFORMATION Processor Q runner is waiting 26 02 03 11 14 12 STATUS There is 1 unprocessed job in the workflow queue 26 02 03 11 14 12 STATUS Running the workflow runner 5 To display results in PLGS click the target plate node The results browser opens 6 As the data is acquired the results in PLGS can be periodically updated by one of the two following methods Click File gt Update or Click amp on the toolbar A 12 Quick Start Tutorials g wl J 5 Container Manager 8 Expression Analysis 5 f g 8 3 S Q z a 3 De Novo Sequencing BLAST Searching Fd a low Designer he Data Preparation ad Modifier Tool 8 n004 Updating project PLGS2Training PLGS with partially acquired sample list Container Manager ED i Ea H Maare tOn iki DIPLGS2Training PROWata Sample_O 3 Processing Parameters EB mataiwr tOM __ Mass spectrum data not yet obtained MaldiPP MaldiWe 2 Oa ___ Mass spectrum data not yet obtained MaldiPP E maw Om ___ Mass spectrum data not yet obtained BE mataipp E mataiwe g AS Mass spectrum data not yet obtained BB MaidiPP E malciwe AG _ Mass spectrum data not yet obtained MaldiPP MaldiWe OA _ Mass spect
285. rmat 1 Select the check box next to the name of the format 2 Click Browse and then select a folder where the spectra output is to be saved If the MS Text Output format is specified the Top most intense peaks to return check box is enabled Selecting the check box enables you to specify the maximum number of peaks written to the MS Text Output file If the check box is not selected the mass intensity pairs of all peaks will be written to the MS Text Output file Plugins tab In PLGS all of the data representing a project gels containers spectra queries results and so on is archived through a supplied PlugIn which saves these projects locally in XML format However it is possible to replace this plugin or add additional third party plugins to handle the project XML ina different manner to parse and write it into a format more suitable for your needs lt Import To save data from other sources and formats into a PLGS project Export To retrieve data from PLGS projects and export the data to other formats 2 23 An example of a PlugIn is the FileSystemPlugIn which is supplied with PLGS This PlugIn is used to import data from other sources into the standard PLGS file structure This PlugIn also exports data from the standard PLGS file structure into other formats For more details of the implementation and use of PlugIns see Appendix C Implementing a plugin for ProteinLynx Global SERVER
286. roject This enables gel spots to be mapped onto plates and viewed in the Container Manager Individual samples can be submitted to MassLynx for automated data acquisition and processing Workflows can be attached to samples for automated Databank Searching AutoMod Analysis BLAST Basic Local Alignment Search Tool Searching and De Novo Sequencing To open the Gel Manager click the Gel Manager icon in the tool tray 9 2 Viewing and processing gel data with Gel Manager Adding and importing data Initially a project needs to be created or opened To create a project see Importing and viewing PLGS sample lists on page 5 3 Adding a new gel without an image 1 2 3 In the navigator tree click the Gels node and then right click Click Add Gel Type a name to associate with the gel in the ProteinLynx browser and then click OK Importing gel spots To import gel spots 1 In the navigator tree click the node of a gel you have created and then right click Click Import Gel Spots Import Gel Spots dialog box Import Gel Spots Plate type Microtitre Target BX 12MICROTITREPLATE X 12 MICROTITRE PLATE OLB file l6 X 8 CAP LC MICROTITRE PLATE e _ Browse oee e Import Gel Spots dialog box parameters Parameter Description Plate type The Plate type onto which gel spots should be mapped Also select the specification of the plate from the drop down list
287. ropped 0 overruns 0 carrier 0 collisions 0 txqueuelen 100 RX bytes 517125 505 0 Kb TX bytes 827 827 0 b Interrupt 11 Base address Oxdf00 Memory feafe000 feafe038 Link encap Local Loopback inet addr 127 0 0 1 Mask 255 0 0 0 UP LOOPBACK RUNNING MTU 16436 Metric 1 RX packets 2980 errors 0 dropped 0 overruns 0 frame 0 TX packets 2980 errors 0 dropped 0 overruns 0 carrier 0 collisions 0 txqueuelen 0 RX bytes 201847 197 1 Kb TX bytes 201847 197 1 Kb root localhost root J The IP address is displayed on the line inet addr 5 Click Next The PLGS Installer program starts Restoring backed up folders If you uninstalled a previous version of PLGS and backed up folders see Backing up the PLGS folders on page 1 7 you should restore them before starting PLGS To do this copy the backed up docs and root folders into the folder where you installed PLGS If you backed up databanks they must be re added to PLGS For details on how to do this see Adding databanks on page 13 3 Running PLGS on Linux To run PLGS you need to start these PLGS modules on each computer Search engine e Microkernel Processor These modules are started automatically when you start the PLGS browser on the machine PLGS can be run from a command prompt or by using the GUI To run PLGS using the command prompt 1 Open a terminal window and then type cd lt PLGS install location gt bin 2 To start the browser type
288. ross the target plate to highlight the spots corresponding to your data files Right click anywhere in the target plate Target Plate pop up menu Select All View Results Merge Results View Sample Information View Attached Templates gt Set Attached Templates gt Set Raw Data File Import Mass Spectrum Process b Plate Settings Click Set Sample to associate the spots with the sample record previously created Select some or all of the spots again right click and then click Set Raw Data File In the Select File dialog box choose the data files to be processed and then click OK Select some or all of the spots again and then right click Click Set Attached Templates gt Processing Parameters Click Choose new Processing Parameters Template from file and then choose the parameter file from disk Requirement To create and alter processing parameters the Data Preparation tool must be used see Getting started with the Data Preparation tool on page 8 2 Select some or all of the spots again right click the click Set Attached Templates gt Workflow Template to RAW data A 3 15 Click Choose new Workflow Template from file and then choose the workflow template from disk Requirement To create and alter workflow parameters the Workflow Designer tool Creating a workflow template on page 7 5 must be used The system is now ready to process and search 16 Select th
289. rum data not yet obtained BE maigipr E maw 2 Ora _ Mass spectrum data not yet obtained BE malaiPP E mawr 2 Oas ___ Mass spectrum data not yet obtained EB mataipr MaldiWF Oat ___ Mass spectrum data not yet obtained MaldiPP g esesesesese BE 38 38 38 98 38 32 38 48 90 898 88 38 38 38 38 38 im Result Summary 00001 11 555 36668 627 6 661 Alcohol dehydrogenase EC 1 1 1 1 For further details on viewing results see Chapter 6 Viewing results in the Results Browser A 13 Acquiring Q Tof MSMS data In this example one sample of hemoglobin digest is used with glu fibrinopeptide B GFP and erythromycin infused by means of LockSpray used as lock mass Setting the microtitre plate To set the microtitre plate 1 Create a new MassLynx project as described in the MassLynx Help 2 Create an MS Method file and LC gradient files in the MassLynx project 3 Create a new PLGS project see Importing and viewing PLGS sample lists on page 5 3 Set the name of the project as Q Tof MSMS 4 Click Container Manager and create a new microtitre plate as described in Creating a new vial microtitre or target plate on page 5 9 Name the microtitre plate Q Tof MSMS 5 Click the plate you have created and then drag over the spot that contains the sample 6 Right click the selected well a
290. s Data Bank SWISSPROT 1 0 gt Minimum peptides 2 l before exclusion MSMS Processing r Digestion m Tolerances Trypsin ed fi 30 Primary Digest Precursor Tolerance ppm Secondary Digest None ps 0 33 Fragment Tolerance Da Databank Searching Missed Cleavages fi r Modifications Fixed Variable Acetyl K Acetyl K Acetyl N TERM Acetyl N TERM Amidation C TERM Amidation C TERM Status Biotin K Biotin K Carbamidomethyl C Carbamidomethyl C Carbamyl K Carbamyl K Carbamyl N TERM Carboxymethyl C C Mannosyl W zj bd Neamidatinn N Carbamyl N TERM Carboxymethyl C C Mannosyl W zj e Neamidatinn N You can change the following parameters Searching parameters Parameter Description Data Bank The Data Bank drop down list will show the available databanks Click the one you wish to search against Digestion Choose the digest reagents you wish to use when searching the data and the number of missed cleavages Peptides Type the minimum number of peptides that must match against a protein before that protein is excluded from further data acquisition Tolerances Type the precursor and fragment ion tolerances to be used by the databank search engine 15 6 Real Time Databank Search
291. s found in the data Therefore due to the possibility of mis assignment the corresponding data is suppressed according to how well the masses match the theoretical masses rather than being completely extinguished Validate Results All MSMS results can be validated A validated peptide will contain a series of three or more consecutive y ions If validation is selected the top scoring peptide for each MSMS spectrum is returned This could increase the requirement for manual validation of the results returned To validate the results select the check box Monoisotopic or Average Rule This attribute applies only to Mascot searches This attribute specifies whether the mass values used in the search are monoisotopic or average In the drop down list click Monoisotopic mass of the first peak in an isotope distribution e Average centroid of the whole isotope distribution Mass Values Rule This attribute applies only to Mascot PMF searches This attribute specifies whether the experimental peptide mass values in a PMF search include the mass of the charge carrying proton MH or if they correspond to neutral values Mr Click the relevant values in the drop down list Peptide Charge Rule This attribute applies only to Mascot Fragment Ion searches This attribute specifies the precursor peptide charge state in a Fragment Ion Search Click the charge state in the drop down list 14 12 Query Tools
292. s on the X axis Show masses on the X axis Copy spectrum data to the clipboard Copy spectrum image to the clipboard a E 2 E S El E View the MSMS spectrum 6 6 Viewing results in the Results Browser Results browser Spectrum viewer toolbar Button Description z View MSMS spectrum ion probabilities Results browser navigator tree The top left component of the results browser is a tree for navigating the workflow results The two different views of the data are protein view and peptide masses view Individual items from the data such as a single protein or mass can be selected within the tree or dragged and dropped from the tree into another component To toggle the navigator tree view click the Protein View and Peptide View buttons below the tree If a workflow contains a BLAST Query then an additional BLAST View is available The BLAST view which is accessible by right clicking the navigator tree and then clicking Show Blast Results does not alter the navigator tree it triggers the display of a BLAST results panel see BLAST results on page 14 26 for further details Protein view The Protein view displays the proteins and ESTs that were matched to the spectrum data by the analyses Proteins and ESTs are grouped into hits each hit represents a set of proteins and ESTs that share the same peptides The following illustration shows a typical Protein vie
293. s that you create However existing templates will use the renderer that was originally applied to that template Setting Automation Setup parameters The configurable parameters in the ProteinLynx Browser Automation Setup dialog box are used by modules that handle automated data acquisition processing and searching To open the ProteinLynx Browser Automation Setup dialog box from the menu bar click Options gt Automation Setup The dialog box has three tabs e Parameters see Parameters tab on page 2 18 enables you to specify the location of modules used in automated processes and alter the behavior of these modules Spectrum Output see Spectrum Output tab on page 2 20 enables you to specify additional formats in which spectra can be saved after processing e Plugins see PlugIns tab on page 2 23 enables you to alter the modules Plugins that handle the archiving and retrieval of ProteinLynx project data Parameters tab A key feature of the ProteinLynx system is its ability to fully automate the acquisition processing and searching of data The Parameters tab enables you to specify the location of modules used in automated processes and alter the behavior of these modules To update the settings click OK 2 18 Setting up ProteinLynx Global SERVER Automation Setup dialog box Parameters tab zi Automation Setup Parameters Spectrum Output Plugins MassLynx Directory P
294. sccssssssessscescsesessccsscsssessscssssssesssssssseseees 3 6 4 Annotating and tracking samples with Sample Manager 4 1 Getting started with Sample Manager cccccccccccccccccccccccceccccccecceeccececs 4 2 Pee AeA les ca auinns Gis eeadereeemn danke 4 2 BYR er o taal sa credasevis ipl aera aa ieee 4 2 Sample editor oniiir RCA ore tr ence etre teres aie eae cee eens te mane ere 4 3 Generating processed samples eessssssssssssssesssosssessosssosssossoosssossossosessossosseseoeos 4 5 5 Specifying samples vials and plates with Container Manager 5 1 What is Container Manager o cccccccsccccccecccccccecccecccecccecccecceecceccceccseceeceees 5 2 Workflow templates and Processing parameters c cscsesessessessseessersneees 5 2 Importing and viewing PLGS sample lists sssssssssssssosssosssossoossoosssossoossoosso 5 3 Taporune PLGP sample ste iic cc acca bein ene iaenen 5 3 Sample eh PAGAL IAI isnie ae Ede need ae ee 5 4 Viewing PLGS sample lists c sc ccsasisssasssaarsvinvssisavevaavvasauvesnasesiarioanaesyiavedeenryenues 5 5 Table of Contents vii Te ECR Wy WNT corse EEA EAN EAA EN AAN E A EAA AE E 5 7 Processing and Searching uccan A A 5 7 aries Tonpa causes csc ae ees a 5 7 Creating a new vial microtitre or target Plate ccccccccccsccscccssccsseseseess 5 9 Settins asample ic sssscdashicees3escvssasteseievedaavede sesnseosasdsauescusecsdasedousvecdesecivadacetsecess 5 11 Attaching raw dat
295. scoring scheme implemented for MSMS searches addresses the question What is the probability that a protein is in the mixture of proteins that constitutes the sample For this reason there can be more than one hit reported as having maximum or near maximum probability of being correct The data consists of a set of mass intensity pairs and their associated uncertainties representing the mono isotopic mass and intensity of every peak in the processed data For each precursor ion a set of peptide sequences is constructed by synthetic digestion of the protein sequences in the database which match within the user defined peptide tolerance of the precursor mass For each peptide sequence the probability of fragment spectrum GIVEN peptide sequence is calculated The natural log of this is the peptide score From these probabilities a list is compiled of the most likely combinations of proteins that could have given rise to the data For example if we have three proteins there are 8 possible combinations The probability of the whole dataset is then calculated given each of these combinations and the probability for a particular protein is accumulated whenever it appears in a combination We assume that the prior probability of each combination is related to the number of proteins in it and use Bayes theorem to calculate the probability of protein present in mixture GIVEN dataset The results are normalized to reflect the number
296. se Shift click to select consecutive species or Ctrl click to select non consecutive species Peptide Tolerance This attribute is optional as a default value is supplied This attribute is used to match intact peptide masses The units used for PLGS searches are parts per million ppm or Daltons Da Mascot searches have additional units available percentage and absolute millimass units mmu The peptide tolerance should reflect the known accuracy of the instrument used to acquire the spectrum data Restricting this attribute to the lowest feasible value can greatly reduce search times and increase the quality of the results 14 6 Query Tools To specify the tolerance type the value into the text field and then click the desired units in the drop down list Fragment Tolerance PLGS or MSMS Tolerance MASCOT Restricting fragment tolerance is encouraged as it can reduce search times Specifying a fragment tolerance is optional as a default tolerance is supplied Rule This attribute cannot be modified for PMF searches as fragmentation spectra are ignored This attribute is used in the final validation of Fragment Ion Search results If the Validate Results attribute is used see Validate Results on page 14 12 this value determines which y ions have been matched successfully It is recommended that this value is set to the lowest value possible but should be at least double the value of the Estimated Calibration Error see
297. shared between them for example if the database of 100 000 entries contained two identical sequences that matched the data more closely than any other candidate sequences the highest scores would approach 10 81 that is 1n 100 000 In 0 5 In this case the ProteinLynx browser would present the proteins as a collapsed hit but in other cases it might not be so easy to judge the effective equivalence of the top scoring matches To uncover minor components in a sample which contained a mixture of proteins it is generally not sufficient to read down the list of top scoring proteins as many of the peptide matches could overlap It is more appropriate to resubmit the data for searching excluding the top hit This effectively down weights data that are matched well by the top hit which allows independent proteins to score highly Other points to consider are As the natural log of values less than 1 results in a negative number very low scores will be reported as negative numbers in the hit list Ifthe protein being analyzed is not represented nor has any homologues in the database the reported scores will be low and of similar magnitude Ifa species specific subset of the database is searched the scores will be expressed relative to the number of proteins in the subset rather than the entire database Scoring Schemes Automatic data curation Depending on the type of search and the search engine used PLGS or MA
298. simple plugin that allows data to be imported and exported from an underlying file system to the ProteinLynx Browser This plugin has thus been termed the FileSystemPlugIn Every time you press the save button in the browser a request is sent to the default plugin to take the associated data and to store it appropriately in the underlying file system Similarly when you select to import data into the browser such as a databank search another request is sent to the plugin to find the associated data within the underlying file system and to return this data to the browser for display The FileSystemPlugIn is the default plugin used within PLGS but you might wish to design and create custom plugins in order to handle PLGS data ina custom manner In order to do this we must further explore the architecture of plugins Implementing a plugin for ProteinLynx Global SERVER Plugin architecture Plugins can be implemented in any programming language that allows access to the standard data streams For example a C language implementation would receive its input through stdin and provide output through stdout Any error messages would be channeled through stderr The integer return value of the main function can also be used to signal the exit status from the plugin see Plugin process exit codes on page C 26 In order to meet user requirements and integrate with third party databases or LIMS systems a plugin interface
299. sion appears in the list of searchable Databanks for each of those tools 13 16 Organizing databanks with the Databank Admin tool Connecting to a search engine The ProteinLynx browser interface communicates with the ProteinLynx Search Engine which regulates Databank searches AutoMod searches De Novo searches and BLAST searches The Search Engine can be present on the local machine Alternatively ProteinLynx browser can be connected to a Search Engine residing on a remote machine Connect to an alternate Search Engine by using the Preferences button Bs and dialog box see Changing preferences on page 2 5 When the procedure has been completed ProteinLynx will connect to the Search Engine on the machine specified Rule Databanks which reside on the local machine and are administered by the local search engine can be viewed searched and edited Databanks which reside on a remote machine and which are administered by a remote Search Engine can be viewed and searched but cannot be edited 13 17 13 18 Organizing databanks with the Databank Admin tool 1 4 Query Tools This chapter outlines the query tools that are available within ProteinLynx Global SERVER By default these tools are not displayed in the tool tray or Tools menu To add the tools follow the instructions in Adding and removing tools on page 2 4 Databank Search tool Enables you to search both MS and MSMS spectra data against a selected databank to identify
300. ssible to select data for multiple wells or spots However only one raw data file can be attached to each well or spot To select more than one well 1 Click and drag around the wells in the Target Plate see Figure titled New target plate display on page 5 10 to import data Specifying samples vials and plates with Container Manager 2 Right click and then click Set Raw Data File Select Files dialog box for multiple files simple Select Files xj Look In E auto01 raw v l e ME Files Selected 0 25 Add z cancer 3 Select the required raw data files in the left hand pane from either the local machine or a remote processor and then click Add To select multiple files hold Shift or Ctrl while clicking 4 Click Advanced to display additional options in which you can specify the workflow and processing parameters templates and also process the data 5 15 Select Files dialog box for multiple files advanced x Look In E autoo1 raw m cas E Files Selected 0 25 Add Del Attach Processing Parameters Template Specifty Template Default MALDI MS lt E Attach Workflow Template Specifty Template Databank search v Ej The dialog box regulates the number of files attached to wells or spots Lamec J poces eee Example If you select nine files and there are six wells only t
301. sting digest reagents cccccccccccccccecceccceecseccscecsecsesecsecceseeseees 12 8 Custom digest reagents ccccccccccccecceccceeccecceccceecceccececsccsecessecseceeseesecsseseseees 12 9 Adding or editing custom digest reagents ccccccsssssesseseesessseseseesereees 12 9 Paving custom digest reagenta i eiiasepaveisenisaidieieeiaimiiendiiaaneaiales 12 10 xii Table of Contents Deleting custom digest reagente sescesicecercereecavseerareereasversasieinaveceatvesauneins 12 10 13 Organizing databanks with the Databank Admin tool 13 1 Getting started with the Databank Admin tool ccccccceecceeeceeeeseees 13 2 Adding Catalans seses eea RES AA EA ER EEE RED 13 3 Databank Arib UTEE asiaa eai 13 4 Editins datapankS isis sessocvacsaddesasvceassccsscscesseasaseueredesazedsueseeacocsuduasucensscsseaceaeese 13 11 Removing and deleting databanks ssessessseesoeeoecoseecoecoeccoecceccececeeccessseee 13 13 Removing databanks from the system record ccsccceeeeeseessteeeeeeeeneeees 13 13 Pilene da aan e eana aS 13 13 Deleting archive MES sostido E E TT 13 14 Deleting revived archive ccccccsesssssscceceecececsesseeeecceceeeeseaeaseuseseeeseeeaeaue 13 14 Keeping archived copies of a databank ccccccccscccseseececeecceeeeeeeeeeeeeeees 13 15 Revive Bir Wehr aA S A 13 15 Connecting to a search engine essssssssssrscccccccccccccccccccecceseececceccesceceseeseecseeese 13 17 IA O
302. t Select the type of search engine you wish to use for this databank search Search Engine Type MASCOT attributes To perform a Databank search 1 Click an attribute in the table see Databank search parameters on page 14 5 for details and then edit the value in the panel at the bottom of the table 2 When the required fields have been edited click the Submit button gt on the toolbar to start the search Databank search parameters The following sections detail the attributes in the Databank Search Parameters table Requirement You must specify the attribute s Search Engine Type Mass Spectrum PLGS and Databanks PLGS or Database MASCOT Search Engine Type You can select PLGS or MASCOT When performing a Mascot PMF search or Mascot Fragment Ion Search select MASCOT from the drop down list Mass Spectrum PLGS or Data File MASCOT This attribute specifies the spectrum data file on which to perform the analysis You can choose a file or URL that contains mass spectrum data To select a file that contains mass spectrum data click File and then choose a mass spectrum file The following formats are valid Mass Spectrum valid data file formats Type of MS data Valid formats MS data MS Text txt XML xml or mzData mzData MSMS data PKL pkl XML xml or mzData mzData To specify a URL click the URL button and then specify or select a URL in the URL Chooser d
303. t Type Default Peptide Tolerance Enter the mass tolerance for peptide matches Peptide Tolerance 50 ppm v 6 Select File gt Save As Name the workflow MALDIWF Attaching the data processing parameters To attach the data processing parameters 1 In Container Manager expand the navigator tree so that the Default MALDI MS node directly below the target plate name is displayed see Adding processing parameters templates on page 5 21 2 Click and then right click the Default MALDI MS node 3 Click Change Processing Parameters A 8 Quick Start Tutorials Processing Parameters Templates dialog box Processing Parameters Templates Select Processing Parameters Template Choose new Processing Parameters Template from file v Default MALDI MS 4 Click Choose new processing parameters template from file and then click OK 5 Click the processing parameter file MALDIPP xml that you created earlier see Setting processing parameters on page A 6 and then click Open Attaching the workflow file To attach the workflow file 1 In Container Manager highlight all the wells on the plate for which you have set samples see Setting the target plate on page A 5 by dragging a rectangle over them Right click 2 Select Set Attached Templates gt Workflow Template to Mass Spectrum 3 Click OK to Choose a new Workflow Template from file 4 Click the MALDI workflow file MA
304. t corner of the ProteinLynx browser 5 27 Tip The same Supertrack spectrum applies to all three replicates of a sample it is not necessary to perform a databank search on the Supertrack spectrum for each replicate To view SuperTrack parameters 1 Click a SuperTrack spectrum node see Workflow and spectrum icons in the navigator tree on page 5 18 in the Container Manager tree and then right click 2 Click View SuperTrack Parameters Result The parameters used for SuperTrack processing are displayed The replicate currently selected in the tree is shown in red To view Supertrack spectra 1 Click a SuperTrack spectrum node in the Container Manager tree and then right click 2 Click View Spectrum Exporting SuperTrack results as XML To export the SuperTrack spectrum as XML 1 Click a SuperTrack spectrum node in the Container Manager tree and then right click 2 Click Export Spectrum 3 Browse to a location and type a name for the XML file to be created 4 Click Save 5 28 Specifying samples vials and plates with Container Manager Interfacing with MassLynx ProteinLynx Global SERVER can export sample lists to MassLynx where data can be acquired The data is then imported back into PLGS where it can be viewed in the results browser Exporting a sample list to MassLynx Once samples are set in PLGS see Setting a sample on page 5 11 but before data is attached to the samples see Attaching raw
305. t have been matched to the selected protein or EST The MS spectrum display colors the peaks matched to peptides from the selected protein or EST The MS MS spectrum display is unchanged Peak mass The protein table is unchanged The peptide table is unchanged The MS spectrum display highlights the peak mass The MS MS spectrum display shows the fragmentation spectrum for the selected peak mass Peptide The protein table shows all proteins and ESTs that belong to the same hit as the peptide and the row showing the protein or EST that is matched to the selected peptide is highlighted The peptide table shows all peptides that have been matched to the same protein or EST as the selection and the row showing the selected peptide is highlighted The MS spectrum display highlights the peak mass that is matched to the peptide The MS MS spectrum display shows the fragmentation spectrum for the peak mass that is matched to the peptide and annotates the spectrum with the peptide fragmentation data 6 10 Viewing results in the Results Browser Items can be dragged and dropped onto other components An example of when this might be useful is when selecting a sequence for a one off AutoMod query PepGrab You can search a selected databank for peptides that match a given mass within a set mass tolerance This enables you to evaluate the quality of a peptide assignment for a given mass and to compare this peptide with others fo
306. t questionable lower scoring hits To open the BLAST Searching tool click the BLAST Searching icon in the tool tray The BLAST Searching Parameters table opens in the editor panel of the browser 14 23 BLAST Searching parameters J Sample Manager Gel Manager V Container Manager Expression Analysis Q Databank Search a BLAST Searching Blast Query Peptide Sequence Databank Numberofits 0 Peptide Sequence Enter the peptide sequence s to be considered for BLAST searching Multiple sequences must be separated by a semi colon only Peptide Sequence i To perform a BLAST search 1 Click an attribute in the table see BLAST search parameters on page 14 24 for details and then edit the value in the panel at the bottom of the table 2 When the required fields have been edited click the Submit button gt on the toolbar to start the search When the analysis is complete the results are displayed in the BLAST results panel see BLAST results on page 14 26 BLAST search parameters The following sections detail the attributes in the BLAST Searching Parameters table 14 24 Query Tools The parameter Databanks is described in Databank search parameters on page 14 5 Peptide sequence In the text box type or paste one or more sequences for searching Each sequence should be a series of amino acid identifiers or a sequence in FASTA format and the seq
307. t software The gel image gel spot container and sample tracking information contained in the file are imported into the current project Progenesis XML file The experiment XML file that can be exported from Progenesis Discovery software The gel image and gel spot information contained in the file are imported into the current project 9 6 Viewing and processing gel data with Gel Manager As part of the import process you must specify the plate names to which the gel spots will be mapped As there is no sample tracking information in files of this type gel spots are assigned to newly created containers in the order they are listed in the file Requirement The gel image file must be in the same directory as the XML file selected 4 Browse to the file and then click Open Replacing the sample in a well or spot To map a microtitre plate well or target plate spot to a different sample 1 In the navigator tree click the Well or Spot node and then right click 2 Click Set Sample Rule The Set Sample option is not available if the current sample has been used to obtain mass spectrum data or workflow results 9 7 Processing data For details of the methods used for processing data see e Chapter 2 Setting up ProteinLynx Global SERVER for details of attaching raw data files workflow templates and processed data Chapter 7 Defining templates for searching with Workflow Designer for details of work
308. tei All Accession ALBU_HUMAN ALBU HUMAN f 69321 i TRFE_HUMAN TRFE_HUMAN P02787 Serotransferrin precursor Siderophilin 76999 nille P02787 Serotransferrin precursor Siderophilin ENO1_YEAST ENO1_YEAST P00924 Enolase 1 EC42111 2phosphoglycera 46642 iile P00924 Enolase 1 EC 42111 2 phosphoglyc GC1_HUMAN GC1 HUMAN P01857 lg gamma 1 chain C region 36083 jile P01857 Ig gamma 1 chain C region KAC_HUMAN KAC_HUMAN P01 834 Ig kappa chain C region 11601 5 k GC2_HUMAN GC2_HUMAN P01859 Ig gamma 2 chain C region 35861 T 2 P01834 Ig kappa chain C region CO3 HUMAN CO3 HUMAN P01024 Complement C3 precursor Contains C3aan 187045 iile P01859 Ig gamma 2 chain C region CLUS_HUMAN CLUS_HUMAN P10909 Clusterin precursor Complement associate 52461 wille P01024 Complement C3 precursor Contains C ALCI HUMAN ALCI HUMAN P01876 lg alpha 1 chain C region 37630 nille P10909 Clusterin precursor Complement asso GC3_HUMAN GC3_HUMAN Po1 860 Ig gamma 3 chain region Heavy chain di 32309 wile P01876 Ig alpha 1 chain C region TNE nille P01860 Ig gamma 3 chain C region Heavy chai A miz z Peakmw Peptide mW Delta Da Delta ppm Probability Log Likelihood Ste wile PO1009 Alpha 1 antitrypsin precursor Alpha 1 pri 674 3418 673 3340 673 3395 0 0055 8 20 15 00 2 2598 a nille P02790 Hemopexin precursor Beta 1B glycopro ae Ser oe me 18 i ie Ca A A B B 2 U f 3 5 Ps WAIE P0142 Ig lambda chain C regions 789 46
309. tein EST table OK Accession l Description mW pl PLGS Score Probability Peptides HBB_BOSGF Hemoglobin beta chain 6 8 5 7 0 28 P02070 HBB_BOVIN Hemoglobin beta chain 15944 7 5 5 7 0 27 P18990 HBB_TURTR Hemoglobin beta chain 16026 6 8 5 2 0 18 P02067 HBB_PIG Hemoglobin beta chain 16024 7 6 5 0 o4 2 15 1 1 When the table is initially displayed or if the Workflow Results icon in the navigator tree is selected the table shows the highest scoring protein or EST from each hit in the results When a hit is selected the table shows all of the proteins or ESTs that belong to the selected hit The following operations can be performed using this table The columns to be displayed the order of columns and the precision with which numbers are shown can be controlled Individual proteins and ESTs can be selected in the table or dragged and dropped into another component Peptide table The middle right component of the results browser is a table that displays a list of peptides Each row in the table represents a single peptide and each column in the table represents a particular data item molecular weight for example The first column in the table indicates whether the peptide match has been set as good OK possible Maybe 2 or poor Not OK B These assignments are either made manually by clicking in the column to cycle through the options or automatically duri
310. tensity Range MALDI MS The intensity range to PSD MX consider MALDI Survey The intensity is specified as a percentage of the maximum possible without saturating the detector Only spectra whose maximum intensity peak above the mass threshold lies within this range will be combined This option is only available when Combine Options is set to Auto select Peptide Filter MSMS Whether to perform background subtraction Background subtraction removes slowly varying low frequency components from the data This can improve the results of subsequent processing Deisotoping and Centroiding attributes Not all attributes are available for all panels check the Applies to column in the table below to see whether the attribute listed relates to the panel you are configuring Deisotoping and Centroiding attributes Attribute Applies to Description Perform Deisotoping MALDI MS Whether to perform Electrospray Survey deisotoping MSMS All three types of deisotoping MALDI Survey simplify the data by replacing PSD MX each ion cluster with a single mass measurement that represents the Carbon 12 peak monoisotopic peak Yes The results are expressed on a singly charged scale No The spectra are peak detected only all isotopes are preserved Deisotoping type All The type of deisotoping to perform slower is more rigorous The three different types of deisotoping are controlled b
311. ter protein 10 12 results 6 2 Expression experiment 10 10 sample annotation 9 10 sample information 5 23 sample lists 5 5 spectrum 5 19 workflows for clusters 10 12 WwW Windows executable file 1 4 installation 1 3 wizard print 6 16 workflow creating A 7 A 17 filters 7 11 for a cluster 10 12 icons 5 18 results 6 10 6 12 templates 5 2 5 6 adding 5 7 5 20 attaching 5 20 printing 11 2 specifying 5 15 Workflow Designer 7 1 7 12 toolbar 7 4 workflow results 6 13 workpad exclude masses 6 31 protein 6 27 X x axis changing the view 6 20 range 6 24 scrolling 6 25 XML 2 20 5 22 14 5 importing project from 3 3 XSL style sheet 7 11 7 12 Z ZIP file importing from 3 3 zoom view 6 25 zooming gel image 9 9 Index 19 Index 20
312. the lower limit of the range click and hold the left or bottom axis slider To modify the upper limit of the range click and hold the right or top axis slider Axis slider Axis slider A 2 Drag the slider to modify the range limit To select data points 1 Click one edge of the area you want to select 2 To select a rectangular area drag to the opposite corner of the area you want to select To select an area freehand hold down Shift while you draw the area you want to select 3 When the correct area is highlighted release the mouse button Result The selected data points are shown in red Click anywhere to deselect the points and start again To perform a databank search on selected data points 2 1 Click the Set Databank Search Parameters button Fa 2 Set the parameters as required see Databank search parameters on page 14 5 for information on the options available 10 18 Using Expression Analysis to compare and analyze sample groups Tip It is advisable to specify a databank that contains the majority of protein sequences that could be in the sample data searched 3 Click amp to close the Databank Search parameters window 4 Click the Search selected items button gt 5 Type a title for the workflow and then click OK Result Protein identifications are returned for the selected EMRTs Searching can take some time progress can be monitored in the bottom right corner of the ProteinLynx browser
313. the plugin and handling the end of the plugin process Implementing a plugin for ProteinLynx Global SERVER Use case the PLGS FileSystemPlugln The FileSystemPlugIn is the default import and export plugin used by PLGS It is used in order to save import data into a PLGS project and also to retrieve data from export a PLGS project held on an underlying file system structure The file system structure consists of the following Root Directory Project Store Project Folder Sample Tracking Folders Workflow Results Folder for Parent Sample Tracking Gels Folder Expression Analyses Folder Expression Analysis Folders Expression Analysis Results Folder for Parent Expression Analysis The FileSystemPlugIn is a Java class plugin it extends the PlugInImp interface This means that the FileSystemPlugIn has 2 distinct methods setProperties and process The setProperties method is used to set specific properties for the FileSystemPlugIn and is called immediately after the FileSystemPlugIn is instantiated The process method is used to process the input output and error messages from the FileSystemPlugIn The input is read from the input stream while the output is written to the output stream and error output is directed to the error stream After the FileSystemPlugIn has been instantiated and its properties have been set it is assigned a PlugInHandler This handler defines how the individual plugin eve
314. this box Requirement At least one replicate must be included for each group 3 Repeat for other groups and replicates When Apply is clicked in this panel the EMRTs Exact Mass Retention Times and Proteins are collated Results A new results node appears below the node for the Expression analysis you are creating For each replicate an icon for the processed spectrum and an icon for the databank search are displayed Click these icons to launch separate windows containing this information Assess Data Quality This section usually becomes important only if you are unsure that the data is of good enough quality to use for quantitation Clicking Apply in the Select Data section takes you directly to Quantitation Analysis The Assess Data Quality section contains a table with a row for each sample group The table contains four columns Group Sample Age and Data View The Data View column contains both a bar chart and a scatter chart icon Click either of these icons to display the Assess Data Quality viewer See Assess Data Quality viewer on page 10 25 for further details Quantitation Analysis Clicking Apply in the Select Data section brings you directly to this section 10 8 Using Expression Analysis to compare and analyze sample groups Depending on the data selected processing can take some time Until processing is complete some options in this section are unavailable The progress of the processing can be
315. this instance the query action is a SELECT and thus a select element has been inserted which describes the type of document to select and what format the returned document should be in In this case the entire document is returned as opposed to a URL of the documents location Implementing a plugin for ProteinLynx Global SERVER In a returned QUERY element a list of references can express the results of the query In the case of large documents usually MASS_SPECTRUM documents containing fragmentation data it can be more efficient to return a URL to the document than to stream the document directly through the plugin The return attribute of the SELECT element allows the client to specify that the plugin return a URL or a reference rather than a document All plugin queries also contain an inner reference element which provide a reference for the query document Reference tags have a single NAME attribute and one or more inner lt REF_ATTRIBUTE gt elements which help describe particular attributes of the referenced document In this case the referenced document is a project that has a PROJECT_ID attribute set to Project 3 The selection of elements of the specified type is predicated upon them having attributes or child elements with attributes matching all those specified by the given reference tree Update of elements lt xml version 1 0 gt lt QUERY gt lt UPDATE ELEMENT_TYPE PROJECT gt lt REFERENCE NAME PR
316. tide matches Digest Fragments enables you to run simulated digests see Running a simulated digest on page 6 29 Bookmark enables you to retrieve the databank entry for the current protein or EST see Retrieving databank entries on page 6 30 Hide Workpad closes the Protein Workpad Coverage map The coverage map shows the protein sequence and a graphical representation of the location of peptide matches The protein sequence is highlighted to indicate the location of peptide matches The color of a highlight depends on the status of the peptide it represents If several peptides cover a particular section of the sequence this section will be a mixture of the highlight colors for the various peptides if they are different in color or a darker shade of the highlight color if the highlights are the same color The highlight colors are explained by a key at the bottom of the coverage map 6 28 Viewing results in the Results Browser Protein Workpad key Key Regions of the protein sequence that match peptides are highlighted in colour according to the key below Matched to a peptide c Matched to a partial peptide E Matched to a modified peptide E Matched to a partial modified peptide E Highlights are transparent so that regions where peptides overlap are visible on the coverage map E g an overlap between a standard peptide and a modified peptide would appear as shown Running a simulate
317. tion void D gt EPN ExecPlugin JavaPlugin ExecPlugIin h PluginHandler execFile File JavaPlugin h PlugInHandler args String workDir File ExecPlugIn className String run void Me ia ae accept v PlugIn Visitor void f properties Properties JavaPlug n getExecFile File run void E l getArgs String accept v PlugIn Visitor void getWorkDir File getClassName String toString String getClassPath URL External Application N PlugInImp setProperties properties Properties void process plugInInputStream InputStream pluginOutputStream OutputStream pluginErrorStream OutputStream int C 27 C 28 Implementing a plugin for ProteinLynx Global SERVER UNIX Help for Installing PLGS on AIX Platforms This section describes using command line input to install PLGS on AIX platforms All changes can be made from the command line In most cases however the more user friendly SYSTEM MANAGEMENT INTERFACE TOOL SMIT can be used SMIT can be invoked from the command line by typing the command SMIT or by clicking on the Common Desktop Environment When possible reference to executing a command through SMIT will be included Contents Topic Page Installing PLGS using the command line D 2 D 1 Hot in y the command D 2 mmand Login as root The login window is either a regular command line window or a C
318. ttached to the data this column is empty View An icon that indicates the status of the data The icon also provides access to the processed spectrum view and the latest workflow results 5 6 Specifying samples vials and plates with Container Manager View column The view column contains an icon indicating the status of the data represented by the row Depending on the status clicking the icon displays the processed spectrum or workflow results View column icons lv Indicates that the data represented by the row has not been processed Lu Indicates that the data represented by the row is processed data or raw data that is newly processed Clicking this icon displays the processed spectrum Rule If the row represents raw data that has been processed several times the processed spectrum displayed is for the most recently processed data Indicates that the data represented by the row has workflow results available Clicking this icon displays the workflow results for the most recently submitted workflow Rule If the row represents raw data that has been processed several times the most recent workflow results for the most recently processed data are displayed Processing and Searching To process and search data from the sample table 1 Click the row representing the data you wish to process To select multiple rows hold Shift or Ctrl while clicking Right click a
319. uences should be separated by semicolons Tip It is possible to drag and drop or copy and paste sequences from the results window of a search that has already been performed Scoring matrix From the list select the scoring matrix for the search The PAM family of matrices were developed by Dayhoff see Dayhoff MO Atlas of Protein Sequence and Structure 5 suppl 3 1978 PAM matrices labeled with low numbers are more suitable for looking for close relationships PAM matrices with higher numbers are more suitable for detecting weaker similarities The BLOSUM family of matrices were developed by Heinikoff and Heinikoff see Henikoff S Henikoff JG Amino acid substitution matrices from protein blocks Proc Natl Acad Sci USA 89 22 10915 9 1992 BLOSUM matrices with high numbers are more suitable for detecting high similarity matches Those with lower numbers are suitable for detecting more distant relationships Results from De Novo searches from mass spectrometry data typically consist of short sequences of the order of 10 30 amino acids When BLAST searching these results it is most appropriate to use parameters which favor short nearly exact matches When searching for short nearly exact matches a preferred matrix is PAM30 The matrix PAM30MS is based on PAM30 but with account taken for the fact that mass spectrometers cannot distinguish between certain pairs of amino acids Expect Threshold Type the requir
320. uery TOOS ss crcasanneincssunccaanenasescsncioidtenssnnsteedsnansacsuetanannecmatecemananntinades 14 1 Query toolbar sisone ae E a E EE EEEN 14 2 Databank Search tool sssissadssasesicassatavscscncisiavsdiassineanecvaniaaaseaacsssiaiviavensaxansiaazes 14 3 Databank search parameters cisnionsinsonienei e 14 5 Search Emrine Type sounen aaa n a Hien 14 5 Mass Spectrum PLGS or Data File MASCOT sssessnsssssssessssssssssesreee 14 5 Databanks PLGS or Database MASCOT esseerserrrerrrerereerrerrserrrerrseesse 14 6 Species PLGS or Taxonomy MASCOT 0 cccccccccccccccccceccseccescesseeseenes 14 6 Peptide Wale ance scccdeicusiiinetinninnianmiaenininn aemmenbadenuaneeas 14 6 Fragment Tolerance PLGS or MSMS Tolerance MASCOT 14 7 Estimated Calibration Error Da Or ppm sssscssesssesssesssssssseesessenens 14 7 Molecular Weight Range PLGS or Protein Mass MASCOT 14 8 ja AG ccs reesei E E AE E O E E EE 14 8 Mimimum Peptides to MAGE cacnistianaianseistnieiee onus winiwars 14 9 Maxim m Hits t Return cc ccsencsecucesstes en E erence 14 9 Primary Digest Reagent PLGS or Enzyme MASCOT ee 14 9 Table of Contents xiii xiv Secondary Digest Roagent cisveisavsisavviveuvereetavadivareeread i wiarieiaeveeere 14 10 Missed CBA aCe sccrives nic iini n NER R RT 14 10 FPized DA a seei ees 14 10 Variable DIO sesia n AS 14 11 Baclade Mieti sece A E 14 11 Ge RESUS cre eddie aces 14 12 Monoisotopic Gr
321. ult 23 B11 Sample_22 digestLMC exp Default CAA 24 B12 Sample_23 digestLMC exp Default Options Acquiring data To acquire data 1 In the main MassLynx window click gt to open the Start Sample List Run dialog box 2 Select Acquire Sample Data and Auto Process Samples 3 Click OK 4 The PeptideAuto Server dialog box opens which monitors the progress of the acquisition MassLynx starts to acquire and process data A 11 Tip The search engine that is active in PLGS when the PeptideAuto window is opened will be the search engine used If you wish to change the search engine close PeptideAuto change the search engine in PLGS and then open PeptideAuto again PeptideAuto Server display Ya PeptideAuto Server OF x Messages 26 02 03 11 14 11 STATUS FiILeSystemPlugIn doing the save now 26 02 03 11 14 11 STATUS FiILeSystemPlugiIn save successful 26 02 03 11 14 12 STATUS FileSystemPlugIn has finished the request was successful 26 02 03 11 14 12 STATUS PlugIn exit code 0 26 02 03 11 14 12 STATUS D PLGS2Training PRO Data Sample 0 has been processed 26 02 03 11 14 12 INFORMATION About to create generator 26 02 03 11 14 12 INFORMATION About to generate pkl and dta output 26 02 03 11 14 12 INFORMATION pkl and dta output generated successfully 26 02 03 11 14 12 INFORMATION Adding item to workflow queue 26 02 03 11 14 12 STATUS Sending D PLGS2Training PRO Data Sample 0 to the wo
322. um Peak Width Perform Deisotoping Choose whether to perform deisotoping All three types of deisotoping simplify the data by replacing each ion cluster with a single mass measurement The results are expressed on a singly charged scale Perform Deisotoping 7 9 Click File gt Save As 10 In the Save As dialog box save with the file name MALDIPP Creating a workflow To create a workflow 1 Click Workflow Designer see Chapter 7 Defining templates for searching with Workflow Designer Click File gt New Select PMF and then click O Right click the Workflow node and then click Add gt Databank Search ee Set the Databank Search Query parameters as shown below A 7 Databank Search Query parameters Workflow Designer Databank Search Query Search Engine Type Attribute m Workflow 22 gt PLGS Workflow Databanks SWISSPROT 1 0 L e Databank Search Query Species Peptide Tolerance 50 ppm Fragment Tolerance 0 1 Da Estimated Calibration Error 0 025 Da Molecular Weight Range pl Range Oto 200000 Da Minimum Peptides to Match Maximum Hits to Return Primary Digest Reagent Secondary Digest Reagent Missed Cleavages Fixed Modifications Variable Modifications Exclude Masses Validate Results Yes Filter None Monoisotopic or Average Monoisotopic MH je Charge 2 Instrumen
323. und in the databank for that mass Tip PepGrab is only available if the databank specified in the workflow template that produced the results was set to Index for PepGrab For details on setting databank attributes see Databank attributes on page 13 4 To use PepGrab 1 Inthe results table click a peptide and then right click 2 Click Perform PepGrab 3 In the list click a databank to search 4 Type a mass tolerance default 0 5 Da 5 Click Search Result A list of peptides that match the mass tolerance is displayed You can scroll through the list and compare the quality of the fragmentation data for each peptide in the list Rule You cannot replace the original peptide assignment with one of the new assignments returned by PepGrab Peptide matches for given mass Results browser 6 11 m PepGrab 951 4531 1 950 4453 Databank SWISSPROT 1 0 Tolerance 0 5 Da Peptide Mass Delta Mass Description DEWDEEE 950 3141 0 1311 Mitotic spindle checkpoint protein MAD2 EDEEETAE 950 3353 0 1099 Eac protein 950 3577 0 0875 Red sensitive opsin Red cone photoreceptor pigmen HMICCCNK 950 3595 0 0857 Elongation factor 1 alpha EF 1 alpha IGEDCDGDK 980 3651 0 0801 Omega agatoxin IID Omega Aga IIID Fragment 950 3651 0 0801 Hypothetical 57 7 kDa protein C40H1 3 in chromosom 950 3699 0 0753 Protein Nimu R1 0 0745 Hypothetical protein IRL10 precursor TRL10 950 3763 0 0689 Zinc finger protein 92 Zfp 92 950 3763 0
324. value is supplied Rule This attribute applies only to PLGS PMF searches This attribute specifies the number of peptides that have to be matched to a sequence before that sequence is considered to be a significant hit The greater the number of matches required for a hit to be returned the more reliable the search results will be However if the spectrum is of poor quality specifying a high value could discount significant sequences In the text field type the minimum number of peptides that a protein must match before it is included in the search results Maximum Hits to Return This attribute is optional as a default value of 20 is supplied Use this attribute to specify the maximum number of hits to be included in the search results It is recommended that you use the default value for a PLGS search of Q Tof MSMS data In the text field type the required number If the search identifies more than the specified number of hits only the top scoring hits are reported Primary Digest Reagent PLGS or Enzyme MASCOT This attribute is optional as a default reagent is supplied The list contains all available digest reagents Click the name of a reagent to select it Rule Only one reagent can be searched at any one time any new selection replaces the existing selection Selecting None or Non specific In addition to a number of pre defined reagents the PLGS menu contains the options None and Non specific None is a suitabl
325. vironment original D 10 UNIX Help for Installing PLGS on AIX Platforms Databanks Formats This section describes the various formats that can be utilized when specifying URLs and using databanks in PLGS Contents Topic Page URL addresses E 2 SPTREMBL flat file format E 3 Genbank flat file format E 6 BLAST flat file format E 8 FASTA flat file format E 9 URL addresses E 2 The URL address Uniform Resource Locator format consists of a Protocol and an Address Examples of possible protocols are http ftp and file To form the URL the address is concatenated onto the protocol name as shown in these examples http www someAddress org filename zip ftp www someOtherAddress org directory flatfile gz file C Directory subdirectory sequences fas Note that URLs are case sensitive Databanks Formats SPTREMBL flat file format The SPTREMBL format is used by Swiss Prot and EMBL Example ID XX AC XX SV XX DT DT XX DE DE XX KW XX OS OC OC OC XX RN RP RA RT RT RL AI304266 standard RNA EST 187 BP AI304266 AI304266 1 03 JUN 1999 03 JUN 1999 IpTRO40 3 EST Rel 59 Created Rel 59 Last updated Version 1 TpTRO40u Channel catfish pituitary library Ictalurus punctatus cDNA clone mRNA sequence Ictalurus punctatus channel catfish Eukaryota Metazoa Chordata Craniata Vertebr
326. w Results browser 6 7 Navigator panel Protein view E Workflow Results ville P41129 60S ribosomal prote wie HNNVIPNGHFK ii nill PF25355 Hypothetical 65 2 k nlll PF00335 Ribitol 2 dehydroge SVLPHLIAQK FAVQAFVHTTR nlll P13743 Llactate dehydroge niil P44166 Hypothetical protei niil P77732 Hypothetical transe niil Q9VWWXZ3 ATP dependent Clp p niil PF10288 Neural cadherin pre wile P04266 Keratin type Il cy The following table details the icons in the Protein and Peptide views Navigator panel icons Protein and Peptide views Icon willlc Description Represents a protein or EST Icons nested directly underneath the Workflow Results icon represent the highest scoring protein or EST for each hit Further proteins and ESTs can be nested within each hit Represents a peak mass from the mass spectrum Represents a peptide Peptides are nested underneath the protein or EST to which the peptide sequence has been matched Represents a peptide with post translational modifications Peptides are nested underneath the protein or EST to which the peptide sequence has been matched 6 8 Viewing results in the Results Browser Peptide view The Peptide view displays masses from the spectrum that were used as queries for the search peptides that were matched to the masses Navigator panel Peptide view Workfl
327. w De Novo Query E Workflow Attribute E Databank Search Query Fragment Tolerance 0 1 Da SN Data Preparation Estimated Calibration Error 0 005 Da Maximum Hits to Return 5 Y Primary Digest Reagent Trypsin Secondary Digest Reagent None Modifications Validate Results Yes C APLGS2 2 docs Filters iDeN Workflow Designer Filter Choose the filter Databank Admin Tool Cd iy Filter CAPLGS2 2 docs Filters DeNovo_filter xsl Digest Reagent Tool File Clear Modifier Tool Enter the filter values in the table below Print Tool Ladder Score Threshold A Q v 3 Mass Threshold Workflow Designer The File button opens a file navigation dialog box which enables you to select an XSL file The XSL file specifies filter parameter names and values which are displayed in the table of filter values The Clear button removes the reference to the XSL file and also the table of filter parameter names and values 7 12 Defining templates for searching with Workflow Designer Creating custom processing parameters The Data Preparation tool enables the creation of custom processing parameters which are attached to raw spectra before processing Contents Topic Page Getting started with the Data Preparation tool 8 2 Attribute sets for data preparation 8 5 Getting started with the Data Preparation tool Processing parameters templates determine how the RA
328. will be helpful to Waters if you request technical support To view log files 1 Go to the directory lt PLGS install location gt log 2 Open the log file in a text editor Processor txt for the processor log SearchEngine txt for the search engine and microkernel log Installation troubleshooting on UNIX 1 20 The following sections detail possible causes and solutions regarding installation problems on UNIX Installer startup problems The installer package can fail to start if there is insufficient temporary space in its current directory To remedy this either run the installer package from another directory or specify the following command line arguments when running the installer PLGS2 2 5 lt unix flavour gt bin is tempdir tmp where tmp is a directory with lots of free space If this does not solve the problem check that the installer package has full permissions by using chmod 777 PLGS2 2 5 lt unix flavour gt bin If the problem persists the file could have been corrupted while being copied from the CD Microkernel failures If the microkernel fails to start check the following e Check that your system is enabled for 64 bit operation this can be done from the smit application If the system is not enabled for 64 bit Installing ProteinLynx Global SERVER operation it might display messages about incorrect libraries when starting the microkernel Check that the permissions levels
329. y different parameters which become available or unavailable depending on the deisotoping type selected Use the slider bar to select slow medium or fast Iterations All The number of iterations 8 12 Creating custom processing parameters Deisotoping and Centroiding attributes Continued Attribute Threshold Centroid Top Applies to All All Description The threshold is a percentage of the area of the most intense peak in the spectrum and is used as a guide to break the spectrum into independent blocks Breaking up the spectrum simplifies the deisotoping problem and speeds up the solution The top percentage of each peak to use to determine its centroid This option is only available if deisotoping is not selected Minimum Peak Width All The minimum peak width Peaks having widths smaller than this number of channels will be removed or merged with adjacent peaks This option is only available if deisotoping is not selected Automatic Thresholds Electrospray Survey MSMS When automatic thresholding is used the deisotoping algorithm attempts to choose a sensible threshold for every spectrum that it is given Although processing the data in this way should give reasonable results experienced users might wish to set thresholds manually to reduce the number of ions reported or to attempt to improve sensitivity 8 13 Deisotoping and Ce
330. y Lock Mass attribute 8 8 Lock Spray Scans attribute 8 8 LockMass tab 16 6 lockspray lock mass 8 8 Log files Linux 1 13 Index 10 UNIX 1 19 Windows 1 6 low energy fragment data 7 5 Low Mass Threshold attribute 8 11 M Make Blastable databank attribute 13 6 MALDI scoring B 4 test procedure A 5 MALDI MS 8 5 MALDI PSD MX 8 5 MALDI Q Tof MS 8 5 MALDI Q Tof MSMS 8 5 processing parameters templates 8 5 Management Options databank attribute 13 7 manually assign samples to groups Expression experiment 10 7 manually define experiment variables Expression experiment 10 6 Manually starting modules on Linux 1 13 on UNIX 1 19 on Windows 1 6 Mascot results 6 5 search engine 7 6 simplifying peaks for 5 26 mass error 6 23 spectrum 14 16 14 21 Mass Accuracy attribute set 8 5 mass spectra 14 5 exporting 5 22 importing 5 22 viewing processed 5 19 mass values 14 12 masses monoisotopic 5 19 Masses to Exclude window 6 34 masses view 6 5 6 7 MassLynx 5 29 Acquisition 15 9 sample list 5 29 A 11 MassLynx Directory parameter 2 19 matrices BLOSUM 14 25 PAM 14 25 scoring 14 25 MaxEnt Lite 15 5 parameter 15 5 maximum hits to return 14 9 substitutions 14 16 Maximum Number of Charges attribute 8 14 Mean Smoothing 8 10 Merge Results parameter 5 23 merging MSMS Spectra 5 24 method file Expression 16 3 MS 16 3 Microkernel Search Engine command 15 15 Minimum Charges to Report attribute 8 14 Minimum Peak Width attribute
331. y provided by the de isotoping software This further contribution can be significant for weak peaks The instrument calibration software provides a Mean Residual To convert this to an estimate of the calibration error it is reeommended that this value is increased by a factor of 1 3 Peak area The importance of a peak is estimated as a function of the signal noise ratio Importance R Area Standard Deviation of Area Where R is a constant that represents the reliability of detected counts This gives a measure of the probability of the peak being real as opposed to representing chemical or instrumental noise The number of matched and unmatched peptides a score is calculated for every peptide in the database The initial prior probability that any given protein in the database is responsible for the submitted data is up or down rated according to these scores The scores are reported as natural logs for presentation purposes Fragmentation data the fragmentation characteristics of peptides at low energy are encoded into a Markov model which incorporates a b y and z immonium ions fragment ions from modifications and internal ions from proline For each peptide sequence the probability of fragment spectrum GIVEN peptide sequence is calculated The natural log of this likelihood is the peptide score Scoring Schemes Search parameters digest reagent with number of missed cleavages fixed and variable
332. y to TRUE if sufficient RAM is available Tip PLGS can read databases from disk Management Options to FALSE Click File gt Save Databank Options The new database is now available for searching from the client PC See also The download location for nrDB is ftp ftp ncbi nlm nih gov blast db FASTA nr Z A 25 A 26 Quick Start Tutorials Scoring Schemes This section introduces you to scoring schemes used by ProteinLynx Global SERVER Contents Topic Page Scoring summary B 2 MALDI scoring PMF PMF fragment ion searches B 4 MSMS scoring fragment ion searches B 5 How do I know if a hit is real B 6 Automatic data curation BF Scoring summary B 2 The factors contributing to the database search scores are The number of entries in the database the correct protein s are assumed to be in the database and the available probability is initially apportioned equally to each entry in the database When comparing calculated peptide or fragment masses with the data it is important to know how well the masses in the data are determined If this estimate is good the information that can be extracted from the data is maximized A good estimate increases the scores of correct identifications An estimate of the precision with which a strong peak can be measured after the instrument is calibrated is the Estimated Calibration Error There is a further contribution to the overall error estimate that is automaticall
333. ysis search parameters AutoMod Search Parameters Attribute Value Sample Manager Spectrum None Peptide Tolerance 100 ppm Fragment Tolerance 0 1Da Estimated Calibration Error 0 005 Da Gel Manager Primary Digest Reagent Non specific Secondary Digest Reagent None eS Missed Cleavages 1 Consider Modifications Yes Container Manager Consider Substitutions Yes Max Mods Subs per Peptide 1 Substitution Likelihood 0 validate Results Yes Expression Analysis Protein Sequences EST Sequences Mass Spectrum Q Databank Search Choose the MassSpectrum XML file Mass Spectrum eoo me ur gear AutoMod Analysis bOO s To perform an AutoMod Analysis search 1 Click an attribute in the table see AutoMod Analysis search parameters on page 14 16 for details and then edit the value in the panel at the bottom of the table 2 When the required fields have been edited click the Submit button gt on the toolbar to start the search When the analysis is complete the results are displayed in the unified results panel that is added to the desktop 14 15 AutoMod Analysis search parameters The following sections detail the attributes in the AutoMod Search Parameters table The attributes Mass Spectrum Peptide Tolerance Fragment Tolerance Estimated Calibration Error Primary Digest Reagent Secondary Digest Reagent Missed Cleavages and Fixed Modifications and Validate

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