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ProHits Analyst User manual

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1. Current user Prohits Administrator Select Project High throughput Projects Select the project to analyze 1 Demo Yeast Gel 2 Demo Yeast Gel Free 3 Demo Human Gel Free Select Lab Name Demo Lab Frequency filter 3 Description Gel free project human From Gingras lab benchmarking set Species Homo sapiens human Permissions Read Insert Add Gel based Sample Modify Delete Upload Search Results Analyst main page When you enter a project within the Analyst module you will see the data workflow and a summary of the icons used in this module The navigator bar on the left lists various visualization and analysis options Fed ProHits Analyst p gi 2 f Protein High throughput Solution E y Sp SO Current user Prohits Administrator search for 2 Database Information Project Demo Human Gel Free ge M Home Database structure Data Management Analyst D nate New En Add Gel free Sample r file conversion Add Gel based Sample search parameters Upload Search Results TPP parameters P Reports Report by Bait ed Re S le a sis etn data parsing Report by Plate i 8 Report by Gel Icons n Modify Click to modify a record F 7 O mun ple Sample Analysis Delete Click LS to delete a record You can only delete your own records d e S C rl pti O n of Comparison Next Level Click to go to next level of a record Picture
2. button at the top of the page and manually enter information Here we are adding a FLAG alone bait Delete Leal Next Level Modify gt Next Bait Report Bait Notes Baits Project Demo Human Gel Free Submit Gel Free Sample Add New Bait Bait List New gene for IP experiment No gene control or non IP experiment O Species Homo sapiens human 5 Name FLAG_alone Epitope Tag N Flag Detail Description Control sample from cells transfected with a vector expressing only the epitope tag no insert Select this option and fill the appropriate info Save To visualize the entry of your new baits in the database go back to the left bar menu and select Report by Bait Delete ea Next Level Modify gt Next ih Bait Report Bait Notes Baits Project Demo Human Gel Free Bait List Column Display Set Experiment status color keys Bait groups select to compa Total Baits 5 1 Page 1 aS ae ee a ee 19 RAFI N Flag 4506401 Prohits Administrator 8 WASL N Flag 51702526 Prohits Administrator t 17 EIF4A2 N Flag 83700235 Prohits Administrator t 6 MEPCE N Flag 47271406 Prohits Administrator ty Total Baits 5 1 Page 1 The Bait Report now lists the baits we have created MEPCE FLAG alone and 3 additional baits that we will use for the demonstration of the functions of ProHits along with some relevant information The ID column lists a unique identifier for the bai
3. Accession NP_062552 2 then dick oas naue GI Number 47271406 P Save Acc Description AF264752_ 1 unknown Homl epere MW 24 95 KDa MVGLDIDSRLIHSARONIRHYLSEELRLPPATLEGDPGAEGEEGTITVRK RSCFPASLTASRGPIAAPOVPLDGADTS FPNNVVFVTGNYVLDRDDLVE Press Save to complete bait entry Sequence AQTPEYDVVLCLSLTKWVHLNWGDB4LKRMFRRIYRHLRPGGILVLEPOP After saving you still have the option to WSSYGKRETLTETIYKNYYRIQLEPEQFSSYLTSPDVGFSSYELVATPHN TSKGFORPVYLFHKARSPSH modify the information a new window appears HAA ass Value ose with two options at the bottom Modify and Next You can add additional information e g in the Description field or modify existing information Hitting Next would bring up the Experimental detail page for this demonstration we will not do this yet Also note that you can create baits for sequences that are not in the database by manually filling in all bold fields species gene name locus tag protein ID protein ID type ProHits does not check for accuracy in these entries You may wish to use this option for example for recombinant or chimeric proteins not corresponding to any of the entries in the database Use the Add New Bait button at the top of the page and continue defining baits in the same manner as for MEPCE Note that in the bait entry page you can also define an experiment in which no gene protein was tagged To do so simply select the No gene control or non IP experiment
4. PSI MI 2 5 This controlled vocabulary drop down keywords can be used for searching and structuring the data using the Advanced Search option Additional annotation Section 3 allows for additional free text annotation in the form of notes Here you can cross reference to notebook page numbers add specifics of the experiment not captured in sections 1 and 2 or describe any problem or deviation from the reference protocols It also allows you to link image files e g Western blots or silver stained gels Navigate through the dropdown menus to select appropriate protocols associated with the experiment Note that selecting the option Edit within Section 2 Controlled Vocabularies of Experimental Details will open up a new window with dropdown menus Controlled Vocabularies Experimental Details Selected Options Click to add new selection or option Edit selection Interaction detection method MI 0007 anti tag coimmunoprecipitation X Cell _ type 293 Fip In T REx Invitrogen Tissue source gt Select all desired fields to capture using the dropdown menus The selected options will be displayed on the right hand side in the order that they were selected Use the Up Down green arrows to change the order or click on the x to remove the entry Select Pass Data to transfer selection to the Experimental Detail page or Close to exit without saving the data Continu
5. radio button and type a name here FLAG_top_contaminants Alternatively append to an existing list by using the dropdown menu Select the row to start importing and check the radio button in the GeneID field Then click Process File Non specific background ras modi set Upload File Field Delimiter V Comma Q Tab Q Space Set Name Add as new FLAG_top_contaminants O Append to existing Selectagroup Transfer first line as attribute name Start Import Row 2 Select EntrezGenelD Field Gene ID O Gene Nam Name O LocusTag O PID SC PT PU C F SF O PID Sc PT PU C bo cw f 4501885 821 94 12 43 70 92 59 100 4501885 1058 113 16 E a Soc ce 203068 russ DAQB 47P19 9 29788785 1246 49 19 51 60 91 53 100 29788785 611 26 10 31 hoses frusa RP13 1228252_ 6174735 1104 43 4 48 10 91 01 100 5174735155903220 3832 KIF11 13699824 1104 38 20 26 90 70 9 75 Ba feen ___asoasoo ceaa7 16 60 30 60 26 75 4502599749321248 y i SUS Once the file is processed the contaminant list will be displayed after selecting the name in the dropdown menu You can manually remove individual entries they will not be on the background list by clicking the delete icon Background Non specific Lists Non specific or background protein datasets are user defined They may consist for example of proteins that adhere to resin in the absence of a bait or of any other proteins tha
6. 55 78 0 00 9 8 MGESDDSILR 2 1121 5 014 01 Viewing results using Cytoscape At the top right corner on the Report page is a link to the molecular interaction visualization program Cytoscape Clicking this link will upload the filtered data with BioGrid interactions if this option is selected Note that all mass spectrometry data will also be uploaded you can use these parameters as attributes of the edges in Cytoscape We will review Cytoscape requirements and basic information in the discussion of the Comparison function Export Sample report Selecting the Export Sample Report on the top right corner allows the user to export text comma separated values CSV or tab separated values TSV files Fields to be exported are user defined and will be exported in the order selected The user can also create pre defined export formats that can be further modified Note that this exports NON FILTERED hits filtered hits can be exported via the comparison tool Export Sample Report Project Gingras_Lab_ Public Alternatively create Sample ID Bait ID Sample MW Sample Intensity a standard format 4675 389 0 000 for reporting Export rows as ZEYEZ Please select columns to be included int Bait M Bait ID Bait Tax ID o D Bait Acc new edit delete Bait Acc Type Bait MW Selected columns B Bait Vector Bait ID 0 Is Gel Free Bait Gene Name Experiment Sample ID 5 Sample
7. Bait Notes callout icon allows you to enter specific notes information for baits or samples Such notes can be a manually entered discussion point Other types of notes include assignment of a project to a user defined bait group Now that you have created baits you are ready to define your experiments Note that in many cases you will be seamlessly going from bait to experiment to sample when entering real samples Here we have simply separated these modules for ease of teaching Creating an experiment gt Return to Add Gel free sample and select start from existing bait This will bring up essentially the same page as shown above but with an additional option green arrow at the extreme right of each row Select this green arrow to enter the experimental details for a given bait The experimental detail page allows you to specify experimental conditions and protocols used for the experiment The top of the page states the bait information below the definition of an experiment can be separated into three sections Delete Lal Next Level Modify Next Experiments Project Demo Human Gel Free Submit Gel Free Sample New Experiment Experiment List Back to Bait Balt Information Bait ID 6 Clone Number PLA Gene ID 56257 Genus Species Homo sapiens human Locus Tag Created 2010 01 05 Gene Name MEPCE Created by Prohits Administrator Family Project Demo Human Gel Free Bait MW 24 950 kDa Status M ES
8. E E E E E T E re 29 Using Cytoscape directly from ProHits comparison ss sssssssrsssnrrrresnnrrrrrnnrnrrrrnnnrrrrnnnrrrrennnrrernnnnrrne 31 Other EXDOre ODUONS teisei iaksani ai e E E E EE EE 32 Comparing larger numbers of baitS sussssnnsssnnrsrnnnnrerenrnrrrrnnnnrrrrunnrerrunnnrrrennnrerrurnrrrrennnrrrrunnrerrune 33 Automatically adding baits for comparison from the baits or sample report list PAGES sssssssssrrssnsrnns 34 se eae ce EEA E S T E E TE AEE E OE OE E EN E ene sawesansereaes 35 Other keywords that can be searched ssssssnssnsnsnnsnnnrnnnnrsnnnrennnrennnrennnrennnrennnrennnrennnrennnrennnrennnnne 37 Manage PrO O O ana e E e e E Oe ee ee 41 TEXU DaSed ProOtOCOlS epaian i aE e T a A a E A E ae 42 PPan a EOT sean EEE SEE eke ENE E EGI EE E E A E 44 Baceno LTS E E A E eens 46 Group LISS rremanen eri e a a e a a a aes 49 EX PORU VESIOLOT 51 ERO De lad EISE a E O 52 Overview The Analyst module allows you to visualise analyze compare search and export your MS results specify project provide analyze results bait experimental compare species details export Bait Protein X Protein Y Analyst Contaminant backup RAW data initiate searches run TPP MS Data management In order to analyze and compare data each MS file in the MS data management module must be linked to a Sample created in the Analyst module For example to create a sample for a gel free experiment y
9. EE EE New Experiment Gel Free Experiment Name MEPCE Biological Material Tet Inducible Fip In 293 January gt 2010 view Section 1 text based Affinity Purification Human cell lysis and FLAG IP GC protocol January See eee protocols view Peptide Preparation Simple in solution trypsin digest of AP samples January ewer eee view LC MS Choose one January view Controlled Vocabularies of Experimental Details Additional Description Section 2 controlled vocabulary Section 3 free text and images western blot images pleage only upload JPG and GIF formatted less than 5 MG image User defined free text protocols In section 1 drop down menus allow for the selection of user specified protocols for each experiment We suggest describing generic protocols in detail in a manner similar to the Methods section of an article The protocols can be entered and managed using the Manage Protocols and Lists option more on this later Controlled vocabulary Section 2 offers via the Experimental Detail Editor the possibility to specify controlled vocabulary to describe the experiment The controlled vocabulary is specified for each project by using the Experimental Editor option within Manage Protocols and Lists Note that this vocabulary can facilitate compliance to community guidelines such as HUPO Proteomics Standard Initiative e g
10. Epitope Tag Lists page 52 This is the only list that applies to the entire local ProHits database The objects in this list are available on the Bait entry page and define the tag if applicable used for tagging of the bait N or C refer to the position of the tag relative to the bait When available the epitope tags have been mapped back to the standard vocabularies from the Molecular Interaction PSI MI 2 5 an automated link to the Ontology Lookup Service OLS We strongly suggest using this service to enter the PSI MI 2 5 terms when entering new tags Text based protocols gt Select the Text based Protocols entry from the Manage Protocols and Lists Protocols Project Demo Human Gel Free Export protocols Biological Material add new import from other projects Affinity Purification add new import from other projects Peptide Preparation add new import from other projects LC MS add new import from other projects Click add new and paste or type your protocol Then press Save Note 1 because the protocols are displayed as html and exported as a CSV or TSV file certain characters and symbols will not display properly and should be spelled out Examples are u u or micro degree and apostrophe Note 2 to each protocol is assigned a unique identifier The protocol can be modified or even deleted as long as it has not been used Once in use modifications are no longer allo
11. Hit Gene Name M Sample ID Sample MW Hit MW Project Frequency O le i Sample Intensity Hit Mascot Score Sample Name C Instrument Raw File Name Unique Peptide Number Total Peptide Number oe Select desired options they Hit Coverage O Hit ID will be listed on the right Hit Description O HitLocus Tag Side and exported in the O Hit Proteinip order selected When done O Redundant Gi select Generate Report M Hit MW lotal Peptide C Result File M Unique Peptide Number O Search Engine M Hit Coverage O Search Database M Hit Description O Filters M Project Frequency Peptide The exported file can be opened with Excel or similar software Using the Notes option Click on the callout icon at the end of any bait row This brings up the following window gt Add desired text and press Save New Notes The following screen can then be seen Only the person who entered the note is allowed to modify or delete it Additional users can create additional comments on the same bait or sample In addition to adding free text annotation default Discussion note type Bait groups Experiment groups or Sample Groups can be created for each project and are managed via the Manage Protocols and Lists option Use the dropdown box to select the desired Notes Types Bait Notes Bait Gene ID 6418 Bait ID 1187 1 Select note type ane BaitGene N
12. Ml 0334 c terminal position OLS Lookup OLSterm flag tag Description N HA ota N TAP EMBL EBI Databases Tools t OLS Home Documentation Project Publications Developer Resources Download Implementation Overview Javadoc Webservice documentation a Contact Us Acknowledgements News y June 2007 Maintenance Release Implementation documentation has been updated to include more recent dependencies Please note that unless stated otherwise in specific instances newer versions of given depencies should adie All Databases Enter Text Here EBI Groups Training Industry About Us Help OLS Ontology Lookup Service Enter Ontology Term Search Ontology Molecular Interaction PSI MI 2 5 MI Be Term Name Include obsolete Ti ID tonne G3 IMi 0s 18 flag tag Additional Information The protein of interest is expressed as a fusion to the definition peptide DYKDDDDKYV for which antibodies are commercially available Sometimes multiple copies of the peptide are fused in tandem Sunset of PSN Alternate label curated by PSI MI FLAG tagged synonym e ee curated by PSI MI FLAG synonym proms provoco curated by PSI MI DYKDDDDKV epitope tag synonym Pai 3665 In addition to the epitope tags currently in the system you can create additional tags by pressing add new and navigating through the fields Again we strongly recommend
13. amplified into 3 x 24 well plates one plate was used for monitoring the expression level tet another 1 after adding tet 1 ug ml for 24 hours and the other well was used to maintain cells Selected clones were amplified in selection medium eventually to 8 x 15cm plates one of them being used for freezing back a low passage stock 4 tubes one for maintaining the culture and 6 for induction and harvesting these were grown without hygromycin prior to harvesting Cells at 60 70 confluence were induced with 1ug mi tetracycline for 24 hours Subconfluent cells 85 95 confluent were harvested as followed medium was drained from the plate 1 ml ice cold PBS was added and the cells were scraped using a silicon cake spatula and transferred to a 15 mi conical tube on ice Cells were collected by centrifugation 5 min 1500 G 4 C the PBS was aspirated and cells were resuspended in 10 mi ice cold PBS prior to centrifugation 5 min 1500 G 4 C This step was repeated once more remaining PBS is aspirated and the weight of the cell pellet is determined Cell pellets are frozen on dry ice and transferred to 80 C until needed Affinity Purification add new import from other projects Peptide Preparation add new import from other projects LC MS add new import from other projects Continue entering protocols as above Alternatively if a protocol of interest already exists in another project to which you have access
14. as a link to the PepXML viewer Experiment Information Experiment ID Name Batch Name Exp Detail Exp Status Inputed by Date Interaction detection method MI 0007 anti tag 4 MEPCE pelletA i rune e naa 369 Prohits Administrator 2010 01 aa Cell type 293 Fip In Peptides from the TransProteomics Pipeline can T REx Invitrogen 7 3 A be filtered based on PeptideProphet scores links Sample Information Besta to the PepXML viewer are also provided Sample ID Sample Code Submitted by 8 MEPCE_pelletA Prohits Administrator POLE Le TT _ oo Ge Hide Filters E ee Fo o o o o o Experiment Filters e aS TPP Probability lt Hyper score lt lon lt Exclude charges 1 _ Mi 2 3 KET HSR ION CHL CHE CHI M ho Exclusion _ Apply Exclusion MascotHits GPM Hits Mascot TPP Hits ER GAT Other TPP Hits Other TPP Peptides Select sample MEPCE_pelletA Total 4278 5 Pages 12 5 a E a e e E n e E 22261 4506679 0 00 6 16 IAIYELLFK 2 1108 65 0 6 22272 4506679 1 0000 63 99 42 34 0 00 36 28 0 00 6 16 AIYELLFK 2 1108 65 044 0 amp 22322 4506699 1 0000 75 39 42 81 0 00 47 67 0 00 8 18 MGESDDSILR 2 1121 5 042 01 22323 4506699 1 0000 96 96 42 81 0 00 5485 0 00 9 18 MGESDDSILR 2 1121 5 0 35 0 6 22326 4506699 1 0000 76 04 4279 0 00 50 13 0 00 8 8 MGESDDSILR 2 1121 5 001 0 amp 22327 4506699 1 0000 97 75 42 71 0 00 53 37 0 00 9 18 MGESDDSILR 2 1121 5 0 07 0 6 22328 4506699 1 0000 97 42 4266 0 00
15. description Opening the Report by plate and clicking the plate icon in the Options field allows you to view your plate layout LJ Available Well 75 Current Band I lt First Plate Ea Previous Plate zy Next Plate En Last Plate FEE Plate MS Completed Plate Plate Report Plates amp Wells Project Demo Yeast Gel Print Preview Plate List Notice In order for Prohits to link raw files automatically please print plate preview to get raw file name formats Modify Plate YDP00155 Plate Layout Prohits Administrator 1 2 3 4 5 6 7 8 9 10 1 12 aaraa SEEE ER A Re e ER a sce ct E a Seen cL IFS ie i it tit 2008 09 19 o Pl me a ea el Pe LL LILLE ete FL EE E EE HL EE S E A S E E ER A a If you wish to use the Auto link option to link your raw files from the Data Management module to the samples in Analyst select Print Preview Plate Information Plate ID 1 Plate Name YDP00155 Created By Prohits Administrator Created On 2008 07 15 15 24 17 Project 1 Demo Yeast Gel Plate Notes Raw file folder Name 20080715_YDP00155_A1_P1 A B c D 3 F G H Bands In This Plate rawsienane username weno caiman sanscase obsonas i species aoi tino moaincaton B06_35 Prohits Administrator 3 P1G1_1P47 G546 jpa BO6 100 000 3 None BO7_36 Prohits Administrator 4 1 IP47 B07 75 000 3 None B08_37 Prohits Administrator 5 P1G1_1P47 G546 jpq B08 60 000 3 N
16. group we reserve the creation of duplicate samples from the same bait experiment for technical replicates e g if we split the final sample in half and run each half separately Note that creating multiple samples from a single bait experiment results in an automatic appending of _A _B etc at the end of the sample name As long as a sample is not linked to any RAW file it can be deleted by the owner Delete ea Next Level Modify Band Sample Project Demo Human Gel Free Submit Gel Free Sample Back to Experiment Bat ID 6 Experiment 4 et Gene ID 56257 Gel Name LocusTag GeneName I MEPCE Gel Image Bait MW 24 950 kDa Method of Staining Clone Number N A Gel Type MEPCE Exp Name MEPCE_pelletA Uploaded Created by Prohits Administrator 2010 01 05 Uploaded by E Band Sample Band Sample aa L Codotname l OPen 1 8 MEPCE_pelletA Now that you have created a new sample entry you are ready to link it to a mass spectrometry raw data file from the Data Management module Links can be created automatically if the nomenclature indicated in the notice below for file naming is respected and ProHits Data Management module is connected to the acquisition computers Alternatively links can be created manually either from the Data Management or the Analyst modules Notice In order to link a raw file to a gel free sample automatically name the folder and raw file as follows e raw file s is in a folder the folder name en
17. on the edge unique peptide N itsmsl pp HEXIME a i d wors hCG 2019956 j b ai EPC ww Y ee ee LARP ji i nal LSM4 PUD FR lt 217272892 y S f q k i RY1 i w A Eog ENDOG 224 136939 gt gt 195972866 lt lletB Edge colour intensity is proportional to spectral counts Note that all of the standard Cytoscape tools are available E prea Recovered bait is circled in red frac cova IF4A 11 EIF4A2 oor coy Experiments Baits are shown as red nodes better Y bg a 2 r IF4G2 Hag 1955 39395 17 rac aio FLAG_alone T Y 194018537 Other export options You may also wish to launch Cytoscape or additional network viewers from an Excel Table in which you can add annotation or other mapping options To do so use the Export table option also located at the top of the table This will create a csv file that can be opened and modified in Excel The file will be displayed as a bait gt hit list with each subsequent column listing a separate parameter These lists are easily opened using a stand alone Cytoscape version gt A B E D E F G H I J K 1 Sample ID Bait Gene Name Hit Gene Name Hit Gene ID Hit Protein ID Hit Score Peptide Number Unique Peptide Number Coverage Frequency Shared Frequency lee Ya 10 EIF4A2_pelletC N Flag EIF4A2 1974 83700235 1759 709 24 71 5 60 20 3 11 EIF4A2_pelletD N F
18. sapiens i 10835063 nucleophosmin 1 isoform 1 Homo sapiens i 4503841 ATP dependent DNA helicase II 70 kDa subunit Homo sapiens i 4502491 complement component 1 q subcomponent binding protein precursor Homo sapiens i 10863889 squamous cell carcinoma antigen recognized by T cells 1 Homo sapiens i 221136939 pre mRNA processing factor 31 homolog Homo sapiens i 4504897 karyopherin alpha 2 Homo sapiens i 23397427 synaptotagmin binding cytoplasmic RNA interacting protein Homo sapiens i 156523968 poly ADP ribose polymerase family member 1 Homo sapiens i 29788785 tubulin beta Homo sapiens gi 10835067 autoantigen La Homo sapiens i 14141152 heterogeneous nuclear ribonucleoprotein M isoform a Homo sapiens i 4506671 ribosomal protein P2 Homo sapiens i 15718687 ribosomal protein S3 Homo sapiens i 167466173 heat shock 70kDa protein 1B Homo sapiens i 5174735 tubulin beta 2 Homo sapiens i 17978466 cyclin Tl Homo sapiens gi 4504445 heterogeneous nuclear ribonucleoprotein Al isoform a Homo sapiens i 4502891 chloride channel nucleotide sensitive 1A Homo sapiens i 157694492 MYB binding protein la isoform 2 Homo sapiens We have now navigated through the table listing the search results However the initial list is not filtered that is all hits including likely contaminants are listed ProHits has a built in filter set that can be applied to the data to help identify bona fide interactors Using fi
19. you can import it directly from that project Click import from other projects select desired project by clicking the gt gt button and pressing Submit Demo Human Gel Free 3 Selected objects Demo Human Gel Free 3 Growth and gal induction for HTP yeast project Protocol ID Protocol Type Project Protecel Mane Created by Creation date Protocol Detail 19 Biological Material Demo Human Gel Free Growth and gal induction for ATP yeast project Prohits Administrator 2010 01 07 Plasmids encoding galactose inducible GAL1 ORF FLAG or GAL1 ORF HA were transformed into the yMT2398 pepddelta strain and a single colony was grown to saturation at 30 degrees C in 1 ml SC medium SC 0 2 amino acid mix 0 5 ammonium sulfate 0 17 yeast nitrogen base lacking leucine supplemented with 0 1 glucose and 2 raffinose An aliquot of this culture was spiked into 340 ml SC leau 0 1 glucose 2 raffinose and cultured for 16 hours at 30 degrees C to reach an OD60O0 of 0 3 0 5 Bait expression was induced for 2 hours by adding galactose to a final concentration of 2 Cultures were harvested by spinning at 5500 rpm at 4 degrees C for 2 min The pellet was resuspended in 1 5 ml of cold harvest buffer 50 mM Hepes pH 7 5 150 mM Na 5 mM EDTA Cell pellets were snap frozen in liquid nitrogen and stored at 80 degrees this yields 2x 0 3 g pallets Tet Inducible Fip in 293 cl
20. you organize your data gt Select the Groups entry from the Manage Protocols and Lists As with the other Protocols and Lists you can define new groups or import a new group from another project Here we will import sample groups from a different project Groups project Demo Human Gel Free Bait Groups add new import from other projects Experiment Groups add new import from other projects Sample Groups add new import from other projects Export Versions add new import from other projects gt To import groups from a different project press import from other projects and navigate through the menus As with the Text based protocols use the green arrows to transfer desired groups to the current project You can only transfer one group at a time Groups from other projects sample Groups Projects Selected objects Demo Yeast Gel Free 2 A Good quality sample ID 10 Name Good quality sample Type Sample Project Demo Yeast Gel Free Description Manually verified sample is good both in terms of bait coverage absence of cross contamination absence of myosin and recovery of interactors Create by Prohits Administrator Initial GS Icon icon_Goodqualitysample gif Ka x Poor quality sample warning ID 11 Name Poor quality sample warning Type Sample Project Demo Yeast Gel Free Description Sample defined by poor bait cover
21. your project for instances of this gene name and indicates matches Click Browse to explore matches This lists all instances of this Gene name across your project Use the Browse buttons to navigate through the data Below we have expanded the Hit Report by Sample option The gene SART3 was identified in both of the MEPCE biological replicates Note that the column Score of Probability Peptides refers to the score from the search engines or TPP and the total number of peptides identified for SART3 in the MEPCE runs Search hits for sart3 Project Demo Human Gel Free Column Display Set X Experiment status color keys Sample groups select to compa E probeniity Opt roba ons Show groups Bait WB Experiment Sample WVersion 4 a j E MEROE Samnita SS MM DREE 1683 242 DE Oe MEPcE palea SOS imne SS MM FREE 1683 105 hE Advanced Search The Advanced Search function can be accessed from the menu bar This function allows you to search for keywords or combinations of keywords and retrieve entries across the following categories Baits Hits Samples Gels Raw Files and Auto Search In the simplest sense you can use the Advanced search in a manner similar to the Simple search i e to retrieve entries associated with a gene name You can use wildcards either at the front at the end or both at the front and end of your query Note that using wildcards especially at the front d
22. 1 M Coverage lt 20 3 V Peptide Unique Peptide lt 2 3 V Frequency gt 25 Em i FLAG_top_contaminant r A ue a i an x V Carry Over E V Spill Over O Auto MW Exclusion s Bio Filters Z C Ribosomal E C Cytoskeleton M C Bait E V Keratin C Artifact Protein F C Translation Elongation Factor E DEAD H Box C Albumin W E 64 Oo 0 191 1 6 6 2 56 d BioGrid overlap NS CO SO AW FQ RP CP BT KT AT TE OB AL C Physical HTP Ww C Physical NON HTP C Genetic HTP Ww O Genetic NON HTP Update Frequency a a 2 Select Apply Exclusion Mascot Hits GPM Hits Mascot TPP Hits Mascot TPP Peptides Other TPP Hits Other TPP Hits TPP Hits Other TPP Peptides 56257 GlyGene MATRI ae 74 310 _bin3 bicoid interacting 3 Homo sapiens 43 80 MEPCE BioGrid a M SCIENCES J 425 4502491 708 C1QBP 686 100 31 349 complement component 1 q subcomponent binding protein 138 9 60 60 GlyGene a M panita precursor Homo sapiens BioGrid nS squamous cell carcinoma antigen recognized by T calls 3 A GlyGene MATRIX gt 413 7661952 9733 SART3 1463 20 109 860 Homo sapiens 105 20 26 90 BioGrid a M SCIENCES 416 55956788 4691 NCL 1011 100 76 570 nucleolin Homo sapiens 84 16 25 80 GliGene MN ee BioGrid se 430 10835063 4869 NPM1 657 100 40353734 32 550 nucleophosmin 1 isoform 1 Homo sapiens 65 7 36 70 GliiGene a My ret FED BioGr
23. 194018537 18 8 195539395 195539395 6 pa 10213 PSMD14 5031981 4 10 56257 MEPCE 47271406 152 184 1 9733 SART3 7661952 105 242 12 51574 LARP 109809739 53 176 13 51691 LSM8 7706425 49 35 14 217272892 217272892 26 30 i15 9129 PRPF3 4758556 26 32 16 221136939 221136939 25 40 17 25804 LSM4 6912486 17 11 18 1025 CDK9 4502747 16 19 2021 ENDOG 53759134 15 34 20 10614 HEXIML 5453682 12 21 57819 LSM2 10863977 8 9 Comparing larger numbers of baits ProHits also allows you to visualize larger numbers of experiments gt Select the baits or samples to be compared and press Generate Report A heat map view of the data will be generated Bait Comparison Color code Hit property color code Shared hits color code O Sortby Total Peptide Number A Bait ID B Descending Ascending O Click to apply filters 8 40 n 111 159 217 283 358 442 Total Peptide Number same as for the normal comparison view with baits SSS E shown as columns and hits EE E m displayed in rows SSS yo pr 3 po u Click anywhere on the I matrix to view the details E E Click anywhere on the map to expand and view names and other details GeneName ProteiniD Links KETAR CCL1 BioGrid 6325282 SGD 5 TFB3 BioGrid 6320668 SGD KIN28 BioGrid 6320095 SGD 2 SSL1 BioGrid 6323033 SGD 2 RAD3 BioGrid 6321019 SGD 3 SCH9 BicGrid 6321
24. 3 WASL_pelletB 8 WASL v 12 WASL_pelletA 8 WASL gu EIF4A2_pelletD 7 EIF4A2 10 EIF4A2_pelletC 7 EIF4A2 9 MEPCE_pelletB 6 MEPCE 8 MEPCE_pelletA 6 MEPCE The selected sample or baits will be automatically added to the Selected Samples and Selected Baits pages of the Comparison view Note that if a bait is selected all samples corresponding to this bait will automatically be added to the comparison view You can keep browsing and adding baits or samples for Comparison as you go These will stay selected for the duration of your session or until you manually remove them from the Comparison page Search options ProHits Analyst allows you to perform simple searches for individual Gene Names or Advanced searches for multiple gene names or keywords in the protein description field or controlled vocabulary Here we will briefly review these options Simple Search Gene name ProHits has a simple search function that is located at the upper corner of the Analyst module main page Enter an official Gene Name then press the right pointing arrow 1 Type a gene name then click the black arrow Fd P ro H its Analyst Protein High throughput Solution Current user Prohits Administrator Bait 0 Hit Report by Bait 1 Browse Hit Report by Sample 2 Browse TPP Hit Report by Bait 5 Browse TPP Hit Report by Sample 5 Browse 0 Sample Raw File Folder total 0 2 ProHits queries
25. 4A2_pelletC SNINWW OO x ERIT EEI 2 Select and transfer the files as above the selected files are now grouped and highlighted with the desired colour E Sampie List o Samples Selected Samples Merge groups BaitID GeneName Tag amplelD SampleName BaitID GeneName Tag SamplelD SampleName new group color E O Control Unmerge o m WASL 12 WASL_pelletA WASL 13 WASL_pelletB RAF1_pelletA RAF1 15 RAF1_pelletB EIF4A2 N Flag 11 EIF4A2_pelletD Cir AA DAL Clan mm ECIiraaAyD mallas D J N OWD wn gt 3 Multiple groups of this type can be created in the same manner If you want to add single files first c select the unmerge button then browse in the file list gt When you are done adding all desired baits and or bait groups press Generate Report This will open a new window the Comparison page Comparison page When you open the Bait Comparison page you will see an unfiltered view of the hits Each column represents a different sample or bait or group of samples or baits if the merge function was used The rows represent each of the hits detected across the n samples or baits Clicking on the Gene Name will take you to NCBI Gene selecting BioGrid will open the BioGrid entry for the given protein clicking on the number in the Protein ID field will bring you to the Entrez Protein page The last column allows you to compare the peptides identified ac
26. 8 Sort Folder Once a raw file has been linked the status bar will display an additional blue icon the number indicates the number of files linked to that entry Baits Project Demo Yeast Gel Free Column Display Set Experiment status color keys select to compare C HA 6319462 Prohits Administrator 19 ALK2 Bait List Bait groups Total Baits 9 1 Page 1 e o F ALK2_sampleA Interaction detection method MI 0007 anti tag coimmunoprecipitation Jj Growth and gal induction for HTP yeast project 2010 1 1 J ProteinA magnetic bead IP for HTP yeast project 2010 1 2 Trypsin digestion on magnetic beads 2010 1 3 J Autosampler LTQ analysis for HTP yeast project 2010 1 4 Jj 2809_ALK2_HA RAW 83 056KB detail Expt Notes List here additional notes e g 1 changes to standard protocol above 2 other experimental details not captured above 3 cross references to internal databases 4 cross references to notebooks 5 data ownership notes F ALK2_sampleB Interaction detection method MI 0007 anti tag coimmunoprecipitation J Growth and gal induction for HTP yeast project 2010 1 2 J ProteinA magnetic bead IP for HTP yeast project 2010 1 3 Trypsin digestion on magnetic beads 2010 1 4 J Autosampler LTQ analysis for HTP yeast project 2010 1 5 E Dink raw file aHa L Expt Notes List here additional notes e g 1 changes to standard protocol above 2 other experimental de
27. 90226 38930 sequences 18115101 residues Timestamp 7 Jan 2010 at 18 45 50 GMT Protein hits gi 40217847 activating signal cointegrator 1 complex subunit 3 like 1 Homo sapiens i 91208426 U5 snRNP specific protein Homo sapiens i 7661952 squamous cell carcinoma antigen recognized by T cells 3 Homo sapiens i 13654237 protein kinase DNA activated catalytic polypeptide isoform 1 Homo sapiens i 47271406 bin3 bicoid interacting 3 Homo sapiens i 55956788 nucleolin Homo sapiens i 217272892 elongation factor Tu GTP binding domain containing 2 isoform a Homo sapiens i 109809739 La ribonucleoprotein domain family member 7 Homo sapiens i 13699824 kinesin family member 11 Homo sapiens i 100913206 DEAH Asp Glu Ala His box polypeptide 9 Homo sapiens i 19923142 karyopherin beta 1 Homo sapiens i 10863945 ATP dependent DNA helicase II Homo sapiens i 5729877 heat shock 70kDa protein 8 isoform 1 Homo sapiens i 50659095 DEAD Asp Glu Ala Asp box polypeptide 21 Homo sapiens i 24431950 PRP4 pre mRNA processing factor 4 homolog Homo sapiens i 4504447 heterogeneous nuclear ribonucleoprotein A2 Bl isoform A2 Homo sapiens a enemy ss tem amen 1914 35 2 1619750 310 322 LAEYQAYIDFEMK 1904 35 2 1620740 310 322 LAEYQAYIDFEMK 1 Deamidated NQ QS i 20070220 protein arginine methyltransferase 5 isoform a Homo sapiens i 4758556 PRP3 pre mRNA processing factor 3 homolog Homo
28. 999 SGD 9996322805 9996321911 9996323454 wS YAP3 BioGrid 6321778 SGD 3 9996324690 man PHO85 BioGrid 6325226 SGD 3 PHO81 BioGrid 6321672 SGD PCL8 BioGrid 6325037 SGD 3 PHO80 BioGrid 6324573 SGD 43 GSY2 BioGrid 6323287 SGD 3 PCL6 BioGrid 6320901 SGD gt Zoom in reveals Baits Hits and selected properties Note however that due to file size the Cytoscape option is not available with this heat map view The Export table option is still available however and can allow you to upload data into a stand alone Cytoscape session the Export matrix function is also available Note that due to large file sizes these export functions may run slowly For additional export functionalities you can go back to the main Analyst module and select the Export Hits option from the left hand menu Automatically adding baits for comparison from the baits or sample report list pages ProHits allows you to select baits or samples to be added to the comparison page while working on other pages To use this option simply click the box located to the left side of each sample in the sample list or by the bait in the bait list Sam ples Project Demo Human Gel Free Column Display Set Experiment status color keys select to compa VM 17 FLAG_alone_pelletD FLAG_alone V 16 FLAG_alone_pelletC 10 FLAG_alone C 15 RAF1_pelletB 9 RAF1 14 RAF1_pelletA 9 RAF1 WV 1
29. Click RJ to have a pop up window to show up and display a gel image S O m e of th e Manage Protocols and Lists Plate Click to view the current plate n y Text based Protocols Next Click to go to next step when submitting samples C O n S u se d n Experimental Editor Co IP Co IP results Yes N Possibi In Progress Bl P Background Lists th IS m O d u le Group Lists Epitope Tag Lists Other Tools Co IP Report Export Functions PPE visualization and analysis options HA Help Data Management Log Out Description of the navigator bar options 1 Create New Entry allows you to define a bait experiment sample and to link mass spectrometry data to this entry These entries can then be linked to specific files in the MS Data Management module Alternatively you can upload search results created by external software 2 Individual Reports allows you to explore your mass spectrometry results Report by Bait provides a list of all baits entered in the database for this project Report by Samples lists all samples entered for this project a bait may be linked to multiple samples especially in gel based projects we also use this nomenclature for technical replicates Report by Plate sample tracking for high throughput projects typically gel based Report by Gel allows you to visualize results for each gel gel based projects only 3 Multiple Sample Analysis Comparison allows you to simultaneously visualize multi
30. Exp Status Inputed by Date Interaction detection method MI 0007 anti tag jess EENE EEI g Prohits Administrator 2010 01 15 Cell type 293 Fip in T REx Invitrogen 32 COPS5 Sample Information Sample ID Sample Code Submitted by 39 COPS5 Prohits Administrator Upload Search Results File Type Otee Omasct Ocpm Select the type of search results files you wish to upload TPP Mascot GPM X Tandem and Browse your local computer for the files in the right format Press Submit to upload search results Upload Search Results File Type tep Onmascot Ocpm Browse TPP Files TPP ProteinProphet select xml file select xml file TPP PeptideProphet Upload max file size 800M Post max size 800M Upload Search Results File Type Otee masco O GPM Browse Mascot Files Mascot File select dat file Filter lons score cut off lt 27 Require bold red peptide v Save Protein score gt save all hits Max number of hits AUTO Significance threshold p lt 0 05 Upload max file size 800M Post max size 800M Upload Search Results File Type Otee Omasco cGpm Browse GPM Files GPM File select xmi file lons expect log e cut off gt save all peptide 0 Save Protein expect log e lt saveall hits 0 Upload max file size 800M Post max size 800M Analyst module 1 19 10 page 41 Manage Protocols and Lists Five types of Protocols and Lists pages are available in Pro
31. Export Functions AA Advanced Search nep Data Management Log Out Bait List Total Baits 9 1 Page 1 NL 6319462 Prohits Administrator J ALK2 paea AA detection method MI 0007 anti tag coimmunoprecipitation Jj Growth and gal induction for HTP yeast project 2010 1 1 J ProteinA magnetic bead IP for HTP yeast project 2010 1 2 Trypsin digestion on magnetic beads 2010 1 3 J Autosampler LTQ analysis for HTP yeast project 2010 1 4 E Dink raw file Expt Notes List here additional notes e g 1 changes to standard protocol above 2 other experimental details not captured above 3 cross references to internal databases 4 cross references to notebooks 5 data ownership notes f ALK2_sampleB Interaction detection method MI 0007 anti tag coimmunoprecipitation J Growth and gal induction for HTP yeast project 2010 1 2 J ProteinA magnetic bead IP for HTP yeast project 2010 1 3 Trypsin digestion on magnetic beads 2010 1 4 J Autosampler LTQ analysis for HTP yeast project 2010 1 5 E Dink raw file J19 ALK2 Expt Notes List here additional notes e g 1 changes to standard protocol above 2 other experimental details not captured above 3 cross references to internal databases 4 cross references to notebooks 5 data ownership notes This brings up a new page that allows you to select the file to be linked to the given entry Wa Note that when you link files from the Analyst mod
32. Fa ProHits Analyst Protein High throughput Solution ProHits Analyst User manual demo Version demo1 prepared on Jan 15 2010 by Anne Claude Gingras with the help of Frank Liu JP Zhang Brian Raught Brett Larsen Wade Dunham Marilyn Goudreault and Karen Colwill Contents VVI N aE EE E A E E E A E elas Phau E ta fhe Bits 2 ACCES 6g 8 0 E AEE ENEE ENEE NE EENE 3 PANY SU THU AGS a a E E E E A E E E AE A 4 Description of the navigator bar OptionSi siasieceostiaccadsqades ns cequcagnsadesiens ca ahactieapedusntenesuss ierti doi ianinri aiat 4 Creating samples and viewing individual reports sssssssssrrrsnrrrsnrrrrnrrrrnrnrrnrnrrnrnrrnnnrrnnnrrnnnrrnnnrrnnnrrnnnene 5 Paanga GOI eG Sari S sister sect ccer cc arenceuacaner car E E E eco eneseses 5 CS IG le E acres eerie eines teem ened eee e EA oe teense E E sec eeendeannen 5 Creating am experime ME vic ncknna diniccan eisd e NE E a E A a a a E aa AE E AE EEE aE E EREE SEAE E aE E RAE aaa aE 8 User dened free text protocols irioiceiriiri iniret inin a 8 CONOCE VOCED NEY eean E E E N E E E E E A E EEE EAE E E EA 8 POROA ANOO a ee E E E scutes E E E A E A A 8 roana NEN SaD E e EEE AEE EEEE E AE E E E E E EE E 11 Linking raw files from the Data Management module s n ssssssusssrnssnrnrrrsnnrrrrrenrnrrrrnnnrrrrnnnnrrrennnnrrrnnnnne 12 Linking raw files directly from the Analyst module alternative s sssssssrsssnrrrrssnrrrrrennrrrrsenrrnrrrnnnnne 12 Novida tng roU Gr tA
33. Gels Project Demo Yeast Gel Submit Gel Sample Add New Gel List Update completed image was successfully uploaded While the image is not mandatory it is highly recommended to link a well annotated image of the Gel Name 1P47 G546 o o gel Method of Staining Coomassie T9 For Project Demo Yeast Gel ea ee n Print Close the Window Uploaded by Prohits Administrator Method of Staining Coomassie Se es ee anes 47 G546 ies shoes N ST1207 ASC1 YST1211 UTE T1127 ILO 42c UTE ST1126 K1 UTE ST786 Y KLO16W UTE Y57659 n o o et La a bt cpc33 ELUTE ELUTE ST1034 YJL149W ELUTE RHOS Gel Image P1G2_IP47 G546 jpg Replace Image ww aso et tem IP run 30 07 01 Gel type TrisGlycine 10 20 Mol Weight 94 35 oe ee n e a a s After a gel is created you can see the information via the Report by Gel function on the left menu R View Gel Image Delete Modify gt Next MS Not Completed MS Completed Gels Project Demo Yeast Gel Submit Gel Sample Add New Gel List Total Gels 1 1 Page 1 C ate net ton 1 IP47 G546 P1G1_IP47 G546 jpg 1 D Prohits Administrator 2008 07 15 A SJ dio gt gt Use the green arrows in the Options field to enter baits from that gel as shown in the gel free sample section gt From each bait define the Experimental Details as shown in the gel free section Clicking on the green arrow in the ex
34. Grid RTI BioGrid PPIH BioGrid LSM4 BioGrid CDK9 BioGrid HEXIM1 BioGrid WDRS7 BioGrid LSM2 BioGrid RPL27 BioGrid LSM6 BioGrid TXNL4A BioGrid LSM7 BioGrid LSM5 BioGrid Overlap is shown in filtered list NHP2L1 BioGrid RY1 BioGrid LSM3 BioGrid WASL BioGrid WIPF2 BioGrid WIPF3 BioGrid WIPF1 BioGrid NCK2 BioGrid RAF1 BioGrid YWHAG BioGrid YWHAQ BioGrid YWHAH BioGrid NRAS BioGrid FKBPS BioGrid KRAS BioGrid MLLT11 BioGrid HAX1 BioGrid EIF4A2 BioGrid PDCD4 BioGrid EIF3J BioGrid EIF4G3 BioGrid EIF4G2 BioGrid w amp P015956 BioGrid Update Frequency 194018537 195539395 5031981 7661952 47271406 109809739 7706425 221136939 53759134 4758556 217272892 10863889 88988289 5454154 6912486 4502747 18959210 122937496 38373695 52630423 4506401 21464101 5803227 4507951 4505451 195972866 4758384 15718761 5802968 13435356 83700235 21735596 83281438 10092601 4503539 GO Cytoscape Export table Export matrix Peptide ing O Using Cytoscape directly from ProHits comparison ProHits allows you to visualize your data using Cytoscape If using the ProHits filters the data post filtering will be displayed changing the filter will modify the display If the BioGrid overlap function has been selected the resulting Cytoscape view will incorporate both your mass spectrometry dat
35. Hits With the exception of the Epitope Tag Lists that are applied to all projects on the local ProHits server the other protocols and lists are only applicable to the current project Lists and Protocols defined for a given project may be imported into a different project so long as the user has access to both projects and permission to modify individual lists and or protocols Access to individual pages of the Manage Protocols and Lists of the Analyst module is restricted via page permissions set in the admin office module We suggest limiting the number of users having access to these management tools Here we will briefly review the function of the different protocols and lists then show a few examples for each category of protocol and or list Text based protocols pages 42 43 Text based protocols provide details on the experimental procedures We have separated the protocols into four modules Biological Material i e what type of cells expression system growth conditions etc Affinity Purification from cell lysis to elution Peptide Preparation including separation at the protein peptide level after elution and LC MS conditions For our internal use we attempt in providing very detailed protocols that could be used for publication with only minor modifications Experimental Editor pages 44 45 The Experimental Editor allows you to create and manage the list of controlled vocabularies to be used within the Exp
36. Hits Other TPP Peptides Update Frequency Search results Mascot Hits 56257 ano GIGene NATRIY E ae 1106 74 310 bin3 bicoid interacting 3 Homo sapiens 43 60 i i SCIEN MEPCE BioGrid E M EJ EJ A complement component 1 q subcomponent binding protein P a E GlyGene MATRI gt 425 4502491 708 C1QBP 686 100 MC E cecairal 138 9 60 60 BioGrid E IM 413 7661952 9733 SART3 1463 20 109 860 one a antigen recognized by T cells 3 105 20 26 90 GlyGene E M a BioGrid _ GIyGene MATRY gt 416 55956788 4691 NCL 1011 100 76 570 nucleolin Homo sapiens 84 16 25 80 2 8 mM ene BioGrid es 430 10835063 4869 NPM1 657 100 40353734 32 550 nucleophosmin 1 isoform 1 Homo sapiens 65 7 36 70 GljGene pura tD BioGrid seminal Search results Towards the bottom of the page are the search results by default these are not filtered The red colour in the ID field indicates the bait as defined by the user when entering the experimental description There are several tabs at the top of the search results table available for navigation The exact tabs displayed depend on the search engines used For the demonstration project we have used the search engines Mascot and X Tandem GPM and have analysed the results using the TransProteomics Pipeline We will first explore the Mascot Hits tab The columns list the following parameters A ID Unique identifier assigned by ProHits for datab
37. Mascot in this example alongside links to initial search results and biological databases Additional export and viewing functions as well as options to filter the hits are also available from this page Over the next several pages we will explore the Results page gt From the sample list page under Options select the graph icon from one of the samples to see the results LJ Exclusion Color RJ Gel image M Mascot GPM Hit Notes Es Yes No Ee Possible E In Progress Sample Report Hits Project Demo Human Gel Free icytoscape Export Sample Report Back to Sample List Bait Information 6 Gl GeneyBioGrid Bait Gene ID 56257 Bait Gene Name MEPCE N Flag Bait Locus Tag Bait MW kDa 24 950 Expo rt and Bait Clone N A Bait Description AF264752_1 unknown Homo sapiens Bait description and visualization Experiment Information Experiment ID Name Batch Name Exp Detail Experimental details Exp Status Inputed by interaction detection method MI 0007 anti g fr tag Prohits T 335 5 oe coimmunoprecipitation Q 1369 Administrator wali el j Cell type 293 Flp In a T REx Invitrogen a Sample Information T Sample ID Sample Code Submitted by o 64 7 8 MEPCE_pelletA Prohits Administrator o0 o9 1 1 0 0 2 0 CO SO AW FO RP CP BT KT AT TE OB AL Use tabs to navigate Show Filters Click to enable filtering options through results pages GPM Hits Mascot TPP Hits Mascot TPP Peptides Other TPP
38. P PeptideProphet XML files Select the Upload Search Results link on the left hand side of the Analyst module This opens up the list of all the baits that you have created in the Analyst module R Gel Image Band in Plate gt Submit sample th Report Upload search results Remove uploaded hits Upload Search Results Project Demo Human Gel Free Total Bands 11 1 Page 1 Bait ID Experiment Exp Gel Lane LaneNum Sample ID Sample Name Uploaded By Uploaded date Uploaded File Options 20 COPS5 39 COPSS5 N Flag COPSS F 10 FLAG_alone N Flag FLAG_alone_pelletC 16 FLAG_alone_pelletC F th FLAG_alone_pelletD 17 FLAG _alone_pelletD F ih 9 RAF1 N Flag RAF1_pelletA 14 RAF1_pelletA 7 D RAF1_pelletB 15 RAF1_pelletB 7 di 8 WASL N Flag WASL_pelletA 12 WASL_pelletA 7 di WASL_pelletB 13 WASL_pelletB 7 D 7 EIF4A2 N Flag EIF4A2_pelletC 10 EIF4A2_pelletc 7 D EIF4A2_pelletD 11 EIF4A2_pelletD 7 D 6 MEPCE N Flag MEPCE_pelletA 8 MEPCE_pelletA 7 di MEPCE_pelletB 9 MEPCE_pelletB ih Total Bands 11 1 Page 1 gt Select the upload option F at the end of the desired sample This pops up a new page Upload Search Results Bait Information 20 GlyGeneyBioGrid Bait Gene ID 10987 Bait Gene Name COPSS N Flag Bait Locus Tag Bait MW kDa 37 560 Bait Clone N A Bait Description Jun activation domain binding protein Homo sapiens Experiment Information Experiment ID Name Batch Name Exp Detail
39. Project User Controlled Vocabularies experimental Deta Click to add new selection or option Edit select yn Interaction detection method HEK293 ATCC 1573 E 293 Fip in T REx Invitrogen x HeLa ATCC cCL2 X Yeast BY4741 EFO_0000098 x Tissue source Background Lists In addition to the Bio Filters and Experimental Filters defined in the Admin Office module ProHits allows you to define additional filters to remove non specific or background proteins These filters are project specific and created within a bait or sample report page in the Analyst module Several different filters can be associated with the same project e g corresponding to different workflows used in the project Creation of these filters requires administrator level privileges The filters can be created by adding proteins manually one by one to an existing list of contaminants The filters can also be generated by uploading a list or table of hits identified in control run s in which case the mapping only requires the Entrez Gene ID field You can also add multiple proteins at once from any other pre existing list e g in Excel The mapping is via the NCBI Entrez Gene ID Select the Background Lists entry from the Manage Protocols and Lists k gt From the entry page click on the modify icon to upload a list of contaminant proteins Background Non specific Lists Non specific or background protei
40. TO UN errn E E A E E A E 15 SRE AMEE OU S E E EE E EE T E AOE E E EO E E EE TE E EE E E T 15 ONINO ODUON e E E E E E EE E E E E E 16 a ga ac Keene a E a ee ee ee ee ee 16 ODEON GOCONNS pieneni a E E de wteecat ee sanssdaniaendsebacete ve niconedewseeeedeniancscobeastuseolan 16 PSG EET eran ee earn es le ge ea ee aetna rats ea eee ene een eet ean 17 Comparing your data to literature interactions 0 ccc cece ee ene eee eee e eee ee eee Sees antennae 19 View and navigate hits from the TransProteOmMics PIPeline ccceccceeee cece e ee eeeeeeeeeeeesaeeeeesaeeeeeaneeenaas 20 Viewing results USING CYtOSCAPC cece cece eee cece eee nee n eee ERED EE EER RESO EEE EERE O OSES ERR EOOSS ESSER REEES EE EERE 21 Export ampe tep OPE aane ace ceoeaaneccossaanvesscanedsensocevdenaececuinn canteceucacuated eanedseucceuedeutesecooseanesoounacian 21 SG TS NORE S OD CONN eaters E gx emacs enee de E E ents dc on eeeetrorentacespecentresmseceotmenss 22 creating gel Dased Sample nirisan ianea a cane ia cccme cer saree EE E a neues lene E cana te ese een eee daenta sie A 23 AGGING a GElDasSed Sample sarcicoveisedvndsaadecovahaid soc taneceuu ssondeartacteden E AEE ehandrelscctedevansecsateantecouaberta 23 The Comparison COO sciececceuscaaveceseaeuercdeaaseietecedineinsecaetennrestdeweweresaareweiescueieteaaeiatdenacesedeaeeecedeaesewetecceineien 26 Mclgeliate Mes POr O COMON OE ttt ter en ie rr er an er ee ee ee ee re ee rr te 27 COMDE OA D e E E E E E EE A EN E
41. a the overlap between your mass spectrometry data and data in BioGrid and data detected only in BioGrid including interactions amongst first neighbours of the hits The colour coding see below allows you to identify the source of the data Before you can use the Cytoscape plug in you need to have the Runtime Environment JRE installed on your local computer you can use the following URL to test whether your computer has a functional JRE http www java com en download help testum xml The first time that you click the Cytoscape icon Cytoscape will be installed on your local computer Press the Cytoscape link immediately above the table to open the current interaction file in Cytoscape The baits are indicated by red nodes alongside the unique bait identifier and the recovery of baits in a purification is indicated by circling the white baits in red The colour coding of the arrows is mapped to the spectral counts as shown above and all peptide annotation is encoded as an edge attribute Note that if the Overlap with BioGrid function has been selected interactions specific to your dataset will be still shown in blue interactions that overlap between your dataset and BioGrid will be shown in green while BioGrid only interactions will be displayed in white The original image is a circular layout in the example shown here this has simply been converted to a spring embedded layout with weight
42. age or overall weaker results than normal samples The data may still be used with caution Create by Prohits Administrator Initial F1 Icon icon_Poorqualitysamplewarning gif Ka X Poor quality sample major ID 12 Upon transfer of a group it will appear on your group list as shown below the new group can be modified or deleted unless it is used for a sample Groups project Demo Human Gel Free Bait Groups add new import from other projects Experiment Groups add new import from other projects Sample Groups add new import from other projects 4 crose contamination major 4 crose contamination warning X Poor quality sample major x Poor quality sample warning V Good quality sample Export Versions add new import from other projects gt To create a new group press add new and navigate through the menus Simply enter a short descriptive name for the group as well as a description an abbreviation that will be listed alongside the baits or samples and an icon Icons can easily be created in Photoshop as 17 x 17 pixel images and saved as GIF PNG or JPEG files A template can be downloaded from the ProHits group page Gr OUPS project Demo Human Gel Free Bait Groups add new import from other projects Name Type Bait Description Abbreviation Max 2 letters icon Please only upload GIF PNG or JPEG for
43. ame SET Bait MW kDa 32 100 Bait Clone N A Bait Description SET nuclearerod 2 Add text Notes Type Notes Close Save New Notes Bait Notes Bait ID 1187 Bait Gene ID 6418 BaitGene Name SET Bait MW kDa 32 100 Bait Clone N A Bait Description SET nuclear oncogene Notes Type Notes Added By Added On Action Anne Claude 2009 03 18 Discussion We should repeat this IP Results are promising Gingras 10 06 48 New Bait Note e Notes Creating gel based samples ProHits has functionality designed to track samples analyzed in a high throughput manner from gel based proteomics Several of the steps are identical to the steps required to create samples for gel free projects Here we will briefly outline the major differences when entering gel based samples Note that you can add samples from in gel digestion as gel free especially if you are only analyzing a few samples without the use of an autosampler Adding a Gel based sample gt Select Add Gel based sample from the left menu and choose whether you will be starting from an existing gel or create a new gel Add Gel based Sample Project Demo Yeast Gel Click this arrow to add gel based sample Link Search EVAS raw file Start from existing gel Se gt To create a new gel add information required in bold and upload the image of the gel RJ View Gel Image Delete Modify gt Next MS Not Completed MS Completed
44. apparent lack of overlap The example bellow shows the effect of the application of a stringent filter on missed interactions Missing interactions Missing interactions STRINGENT FILTER NO FILTER BioGrid interactions not found Show found hits RERE BioGrid interactions not found O Show found hits ES Bait Gene Name FAM40A Gene BioGrid Bait ID 389 Bait Gene ID 85369 4163 MCC GeneyBioGrid Bait Gene Name FAM40A Gene BioGrid 5515 PPP2CA GeneyBioGrid 4 4163 MCC Gene BioGrid w 5516 PPP2CB GeneyBioGrid A 5516 PPP2CB GeneyBioGrid 4 5518 PPP2R1A GeneyBioGrid A 10494 STK25 GeneyBioGrid 4 10494 STK25 GeneyBioGrid A 80342 TRAFSIP3 GeneyBioGrid A 11235 PDCD10 GeneyBioGrid 4 29888 STRN4 GeneyBioGrid 4 57464 FAM40B GeneyBioGrid 4 80342 TRAF3IP3 GeneyBioGrid A View and navigate hits from the TransProteomics Pipeline The tabs located immediately above the search results table allow you to explore search results that have been parsed from the PeptideProphet and ProteinProphet components of the TPP In the page Mascot TPP hits different filtering options based on the number of unique or total peptides as well as the probability values for the TPP have been implemented A link to the TPP search result viewer is provided in the Option column of the table orange Institute for Systems Biology icon this opens up the standard ProteinProphet view allowing further exploration of the data Hid
45. ase purposes B Protein Protein accession number from original database used by the search engine C Gene NCBI Gene ID Gene Symbol mapped by ProHits from Protein accession D Score Mascot score if applicable E Expect value GPM X Tandem Expect value if applicable F Frequency The frequency that this protein hit is detected across all samples analyzed for this project G Redundant Other protein accession numbers matching the same set of peptides H MW kDa Calculated MW for the protein I Description Definition field from the NCBI protein entry J Peptide Spectral counts or total peptides as calculated by the search engine K Unique Peptide Number of unique peptides as calculated by the search engine L Coverage Percentage of the indicated amino acid sequence identified by your search engine M Links External links to the NCBI Entrez Protein page GI the NCBI Gene Page Gene and the BioGrid BioGrid N Filter provides a colour coded view of the Experimental Filters or Bio Filters that could be applied to remove each hit O Option Provides the list of peptides belonging to this hit green M icon opens up the original search engine search results here Matrix Science icon for Mascot search results and allows for the addition of Notes call out icons includes manual exclusion Sorting options You can sort the results from any of the black underlined columns Score Peptide UniquePeptide and Covera
46. ate a new sample from an existing bait or 2 create a new bait Here we will start by creating 5 new baits for this project Creating a bait gt Select the Add Gel free Sample link under Create New Entry Select new bait from the dropdown menu then click on the Bait Blue arrow Fa Pro H its Analyst Protein High throughput Solution Current user Prohits Administrator Add Gel free Sample Project Demo Human Gel Free Click this arrow to add gel free sample Search raw file Add Gel free Sample Add Gel based Sample Upload Search Results il Individual Reports Start from new bait 7 This will open a new page Note at the top of the page the data structure the Bait is highlighted indicating that you are adding bait level entries Note that each of the baits is automatically assigned a unique numeric identifier The fields highlighted in bold indicate that the information is mandatory but many of these can be filled automatically The easiest way to enter a new bait is to simply 1 select the desired species here we have selected Homo Sapiens 2 enter an official Gene Name HUGO for human here we selected MEPCE 3 click the Get Protein Info green button Clicking Get Protein Info automatically retrieves the protein information which is displayed in a new window Verify this information and hit Pass Value if correct the information will automatically be t
47. ds with the Project ID e start with sample ID and first 4 characters of sample code AnyNameNoSpace_P3 project ID is 3 D 8_MEPC_Any0OtherWordNoSpace RAW Linking raw files to a created sample Linking raw files from the Data Management module gt From any page in the Analyst module click Data Management on the left menu bar shown by orange arrow on the right link the desired file as described in the Data Management section parse the hits and return to the Analyst module Linking raw files directly from the Analyst module alternative For this alternative example we are linking files from the Demo Yeast Gel free project which you can access by going back to the home page of the Analyst module Go to the Report by Bait or Report by Sample page of the Analyst module and click on the Status column of the desired file to display experimental details Select Link raw file Baits Project Demo Yeast Gel Free Column Display Set Experiment status color keys Bait groups select to compa PF rome D create New Ent Add Gel free Sample Add Gel based Sample Upload Search Results Bal pnctvicua Reports Report by Bait Report by Sample Report by Plate Report by Gel D uito Sample Analysis Comparison Ef Manage Protocols and Lists Text based Protocols Experimental Editor Background Lists Group Lists Epitope Tag Lists S Other Tools Co IP Report
48. e Filters mia eee cana Viewing the Hits From TPP allows you to Vi TPP Probability lt 0 9 M Coverage lt 20 V UniquePeptide lt 2 Total Peptide lt Frequency gt 452 il filter or sort based on TPP probability values Bio Filters and to open the ProteinProphet page C Ribosomal W C Cytoskeleton W C Bait E V Keratin Artifact Protein M C Translation Elongation Factor E DEAD H Box C Albumin W B o Oo No 0 PBT COV UPT TPT FONpP CP BT KT AT TE DB AL BioGrid overlap O Physical HTP Y C Physical NON HTP O Genetic HTP 1 O Genetic NON HTP A No Exclusion Update Frequency Mascot Hits GPM Hits MMERCamig ancaM Mascot TPP Peptides Other TPP Hits Other TPP Peptides Pet IDs E pose 3 55 20 bin3 bicoid interacting 3 Homo sapiens 54 00 IGIGene M Gc MEPCE BioGrid 2663 7661952 9 33 1 0000 2 43 20 squamous cell carcinoma antigen recognized by T cells 3 Homo sapiens 131 40 28 70 GDGene Me SART3 BioGrid 51574 GlyGene 2522 109809739 1 0000 1 88 40 109809741 La ribonucleoprotein domain family member 7 Homo sapiens 86 25 31 30 i i miei BioGrid M 6 2666 7706425 yee r 1 0000 1 15 20 U6 snRNA associated Sm like protein LSm8 Homo sapiens 56 14 80 20 GlyGene M E BioGrid The Mascot TPP Peptides tab lists all of the parsed parameters at the peptide level and provides some basic filtering options as well
49. e filling experimental details link any desired image and press Save Delete fia Next Level Modify gt Next Experiments Project Demo Human Gel Free Submit Gel Free Sample New Experiment Experiment List Back to Bait Bait Information Bait ID 6 Clone Number NJA Gene ID 56257 Genus Species Homo sapiens human Locus Tag Created 2010 01 05 Gene Name MEPCE Created by Prohits Administrator Family Project Demo Human Gel Free Bait MW 24 950 kDa Status DHI I Update completed no new image uploaded Experimental Details Experiment Name MEPCE Biological Material Tet Inducible Fip in 293 2010 1 1 change view Affinity Purification Human cell lysis and FLAG IP GC protocol 2010 1 2 change view Peptide Preparation Simple in solution trypsin digest of AP samples 2010 1 3 change view LC MS Pressure bomb load LC MS on LTQ BL protocol 2010 1 4 change view Controlled Vocabularies of Experimental Details Interaction detection method Ml 0007 anti tag coimmunoprecipitation Cell type 293 Fip In T REx Invitrogen Additional Description ist here additional notes e g 1 changes to standard protocol above 2 other experimental details not captured above 3 cross references to internal databases 4 cross references to notebooks 5 data ownership notes Images attach image western blot images please only upload JPG and GIF formatted less than 5 MG image Creat
50. e the order of the columns in the Comparison View Use the green up down arrows on the right hand side to reorganize the sort order Individual Baits or Groups of Baits can be reorganized F 5 Sample List Samples Selected Samples Merge groups BaitID GeneName Tag Sample D SampleName BaitID GeneName Tag SamplelD SampleName new group color pay 10 FLAG_alone N Flag 17 FLAG_alone_pelletD WASL_pelletA O Control 10 FLAG_alone N Flag 16 FLAG_alone_pelletC WASL_pelletB 7 EIF4A2 N Flag 11 EIF4A2_pelletD RAF1_pelletA Unmerge 7 EIF4A2 N Flag 10 EIF4A2_pelletC RAF1_pelletB MEPCE N Flag 9 MEPCE_pelletB MEPCE N Flag 8 MEPCE_pelletA Use up down arrows to reorganize Sort by group type Bait O Sample the selected baits for Comparison Select Samples s by clicking then press the gt gt key Selected Samples are displayed on the right side Merging files prior to Comparison Additional options are available that provide merging options for two or more files Please note that the merging is a very simple process that simply reports the best hits for the item but does not do any recalculation If the selected display option in the report is the Mascot score the best scoring hit will be listed if the selected display option is based on spectral counts the hit with the highest spectral counts will be reported The merging function allows you to group two or more control runs click on the Control butto
51. east one of the words option to search for different keywords even across different fields This will generate a list of results that will be the union of the separate lists Hits searches returning too many results Note that there is a limit of 3000 to search results Try narrowing down your search parameters and try again Example Searching for squamous AND carcinoma in 293 Flp In T REx cells and in anti tag coimmunoprecipitation date restricted to January 2009 January 2010 Advanced Search Project Demo Human Gel Free instructions Word s or value s to query squamous carcinoma Add wildcard attheend atthe front front and end no wildcard Find at least one of the words separated by a space character all words separated by a space character the exact phrase V include description Experiment Detail Interaction detection method MI 0007 anti tag coimmunoprecipitation AND Cell type 293 Fip In T REx Invitrogen select remove 2009 01 To 2010 01 select remove Search O T Uploading search results The Analyst module allows you to import search results from the TransProteomics Pipeline TPP Mascot or GPM X Tandem This function is very useful for laboratories that are not interested in the Data Management module of ProHits e g if they are using a third party analysis solution All that is needed for this section are the search results files or both TPP ProteinProphet and TP
52. ecreases search speed Specify keyword s to be searched Word s or value s to query Add wildcard atthe end atthe front frontandend no wildcard O Use wildcards at least one of the words separated by a space character O all words separated by a space character Search or limit searches using controlled vocabulary for Perform logical operations the exact phrase D include description Experimental Details Also search in the Description fields Experiment Detail select remove Date select remove Limit searches by date Search This will return a list of results that you can then explore further by selecting the Browse option for each of the categories as for the simple search Other keywords that can be searched In addition to the Gene Name different keywords can be searched The fields searched depend upon the category as defined below 1 Bait the keywords were detected in the entry for a bait fields searched are Gene Name Gene ID Locus Tag Protein ID Epitope Tag Bait Mutation Clone Number Vector with optionally Bait Description The searched fields are indicated by red ovals below Baits Project Demo Human Gel Free Bait ID 6 i j Species Homo sapiens human If the bait sequence has been modified altered you can first enter the wild type gene name to g
53. ed by Prohits Administrator c e Upon saving you will be given the option to Modify the entry or follow the green arrow to the next page to enter specific samples Additionally you can continue creating experiments by toggling between the New Experiment Experiment List and Back to Bait buttons at the top of the page to enter biological replicates for each of the baits Return periodically to the Back to Bait list to monitor your progress Note the colour coded experimental status bars in the table This view shows our five baits with experiments defined for four of them MEPCE EIF4A2 WASL and RAF1 The status column displays experimental details experimental status and bait groups see below The colour coding in the Status column indicates that information has been entered for each of the specified fields gt Click on the colour coded status bar to obtain additional experimental details In the Bait view experiments and samples defined under the same bait will be combined in the same row multiple experiments will be shown by stacked colour bars Note that you cannot delete baits for which experiments have been defined note in the picture below that the FLAG_alone bait can still be deleted since no experimental details have been entered yet Start by deleting the Experimental Details and work your way up as previously described Delete ial Next Level Modify gt Next th Bait Report EJ Bait Notes Ba
54. erimental Details page in conjunction with the text based protocols and additional notes For our internal use we attempt to capture information that would allow PSI MI 2 5 compliance as well as other relevant information that would allow us to structure our data Note that the terms entered in this section are searchable in the Advanced Search function Background Lists pages 46 48 This function allows you to define and manage one or more lists of contaminants and or background proteins associated with a given project For example you could maintain individual lists of the proteins found to associate non specifically with different affinity matrices The proteins on a given Background list can be subtracted from the list of identified proteins in Individual Report Comparison or Export views Group Lists pages 49 51 This function allows you to further organize and or mark certain baits experiments or samples by adding a colour coded and user defined icon that will appear in the Status bar of the Report by Bait or Report by Sample view Useful Sample level group could include comments about the quality of the data while Experiment level group would refer to some property of the experimental prep e g phospho enrichment and a Bait level group could be the type of tag used Additionally ProHits allows you to mark at the Sample level samples that are to be included in publication and or to be exported to a third party
55. erlap BioGrid interactions not found here a ee ee ee POSL NS CO SO AW FQ RP CP BT KT AT TE OB AL O Physical HTP Y C Physical NON HTPA 9 Genetic HTP Y7 O Genetic NON HTP A Mascot Hits GPM Hits Mascot TPP Hits Mascot TPP Peptides Other TPP Hits Other TPP Peptides 56257 GI Gene meme ae 1106 74 310 bin3 bicoid interacting 3 Homo sapiens 43 80 MEPCE BioGridy M feens ED 413 7661952 9733 1463 20 109 8609 S4Uamous cell carcinoma antigen recognized by T cells 3 Homo 105 20 26 90 IGIGene puna Fea E SART3 sapiens BioGrid 419 109809739 npr 955 20 109809741 66 860 La ribonucleoprotein domain family member 7 Homo sapiens 53 14 25 80 IGIGene mM n ED BioGrid 468 7706425 panaki 305 20 10 400 U6 snRNA associated Sm like protein LSm8 Homo sapiens 49 3 61 50 GlyGene punn ta LSM8 BioGrid Note that the graph on the right indicates the number of hits that have not been filtered out but belong to the different categories that could be filtered out In this example after filtering only 1 RP Ribosomal Protein remains as compared to 64 in the unfiltered example Comparing your data to literature interactions Prohits allows you to automatically query the BioGrid interaction database for previously reported interactions specific to your bait To do so select the type of interactions desired physical interactions from high throughput HTP studies physical interactions not from HTP studies non HTP genet
56. et its protein information then modify the Gene Name as you wish and write the modification detail in Description feld Get Protein Info GenelD f 56257 Lo ocusTag This feld 1s ignored if a Gene ID ts specifed when you click Get Frotein Info button ProteinID 47271406 IE ProteiniD Type Gil MW 24 950 kDa Family Clone Number N A Created by Prohits Adminisirator Created time 2010 01 05 Description 2 Hits the keywords were detected in the hits list field searched is Gene Name with optionally Protein Description You can similarly see the hits across TPP results The searched fields are indicated by red ovals below Mascot Hits ear 56257 eT bing bicoid interacting 3 Home ie AT27 1406 MEPCE 1106 20 74 310 canlane 425 4502491 708 CiOBP 686 34 1 J THES squamous cell carcinoma antigen 4135 7661952 9733 SARTS 1463 20 105 86 recognized by T cells 3 Homo 3 Sample the keywords were detected in the user defined Sample Name 4 Gel the keywords were detected in the fields Gene Name Gene Image and Lane Code 5 Raw files the keywords were detected in File Name or Folder Name This brings you to the Data management module and lists the folders files bearing the selected keywords 6 Auto Search the keywords were detected in Search Task Name This brings you to the Data managemen
57. ge sorting can be in ascending or descending value Links details The following pages can be obtained from each of the items in the Links column seeeee 30 ey My NCBI a ee 000 eo Sian in Reaisted Search Protein for Limits Preview Index History Clipboard_ _Details gt Format GenPept FASTA Graphics More For My NCBI a Entrez Gene lt Sian in east NCBI Reference Sequence NP_055521 1 squamous cell carcinoma antige Scarch Gene for Homo sapiens Search Limits Preview Index History Clipboard Details a La ed Display Fuil Report 3 Sendto General Repository for Interaction Dataset Locus NP_055521 4 home help support contribute downloads mirrors about us DEFINITION squamous cell carcinoma ar sapiens J 1 SART3 squamous cell carcinoma antigen recognized by T cells 3 Homa ACCESSION NP_055521 i VERSION NP_055521 1 GI 7661952 Genel 2722 Homo sapiens DBSOURCE REFSEQ accession NM 0147 aaam A I7 Aliases MGC138188 RP11 13G14 P100 p110 p110 nrb DSAP1 KIAA0156 TIP110 SOURCE Homo sapiens human J ORGANISM Homo sapiens Official Symbol SART3 z D Description squamous cell carcinoma antigen recognized by T celis 3 Eukaryota Metazoa Chord k gt u emat Pag oie pease Official Full Name squamous cell carcinoma antigen recognized by T cells 3 4 a si Links UniprotKB OMIM HPRD Entrez Gene atarr
58. he same Alternatively multiple samples from one experiment may be created when the sample has been fractionated e g by strong cation exchange prior to the analysis Each of the fractions is then assigned a different Sample name within the same experiment The Notes sections from the Experimental Details page should explain the sample naming scheme Note that we enter biological replicates as different experiments from the same bait Creating a new sample Following the green arrow on any of the Experimental Details pages will open a new window allowing you to create one or many samples for a given experiment gt In the Sample page select the Add New Sample button to create a sample entry for this bait and set of experimental conditions Delete a Next Level Modify Band Sample Project Demo Human Gel Free Submit Gel Free Sample Back to Experiment Bait ID 6 Experiment 4 C Gene ID 56257 Gel Name LocusTag GeneName I MEPCE Gel Image Bait MW 24 950 kDa Method of Staining Clone Number N A Gel Type MEPCE Exp Name MEPCE_pelletA Uploaded Created by Prohits Administrator 2010 01 05 Uploaded by a Add New Sample Band Sample Band Sample By default ProHits will use the experiment name to name the first sample created from the relevant experiment ProHits will also assign a unique Sample ID The sample name can be modified if necessary in this case just type the desired sample name in the text box In our
59. hini Hominidae Hon a REFERENCE 1 residues 1 to 963 3 AUTHORS Mohamed E R Naito M Te Primary source HGNC 16860 Gene Ontology 1 total GO annotations for this record view list Shichijo S Itoh K and t TITLE Capability of SART3 109 11 See related Ensembl ENSG00000075856 HPRD 11532 MIM 61168 Download interactions associated with SART3 lymphocytes from prostate A31 and A33 alleles Gene type protein coding JOURNAL Int J Oncol 34 2 52 PUBMED 19148489 RefSeq status REVIEWED REMARK GeneRIF Levels of anti s Organism Homo sapiens patients are significantly subjects Lineage Eukaryota Metazoa Chordata Craniata Vertebrata Eu REFERENCE 2 residues 1 to 963 Mammalia Eutheria Euarchontoglires Primates Haplor AUTHORS Licht K Medenbach J Li Catarrhini Hominidae Homo TITLE 3 cyclic phosphorylation Displaying ai Data recycling factor pll10 thre Also known as P100 p110 DSAP1 TIP110 KIAA0156 MGC138188 p JOURNAL RNA 14 8 1532 1538 20 RP11 13G14 SART3 PUBMED 18567812 Ld Aliases Description Evidence Code Role Source interactions REMARK GeneRIF Both purified anc Summary The protein encoded by this gene is an RNA binding nuch recruit pll0 protein to U is a tumor rejection antigen This antigen possesses tum RNA binding proram 1 serne ncn ooman Sari rert e a sagas capable of inducing HLA A24 restricted and tumor specifi ESI Gene Ontology Vew List AUTHORS Matsuoka S Ballif B A lymphocytes in cancer patie
60. ic interactions of both types and press Apply exclusion The interactions that overlap with the literature will be highlighted in the filter column Note that the definition of HTP and non HTP is from BioGrid high throughput papers are identified as such by BioGrid curators as a default publications reporting gt 100 interactions are also identified as HTP Follow this link to see interactions 1 Select desired type s of data to visualize reported in BioGrid but not detected here BioGrid overlap BioGrid interactions not found here M Physical HTP w M physical NON HTP M Genetic HTP YI M Genetic NON HTP A No Exclusion pe 2 Select Apply Exclusion ares TEA interactions Hits From Search Engines Hits From TPP Peptides From TPP 85369 GI Gene puran FED ae FAM40A 2301 11 88 96 260 hypothetical protein LOC85369 Homo sapiens 39 52 90 BioGrid a SCIENCES fe 86113 142976686 29966 STRN3 1588 14 38 87 550 nuclear autoantigen isoform 1 Homo sapiens 73 24 41 80 steht ry A M rE l 1 86112 51242945 6801 STRN 1891 13 75 86 540 striatin calmodulin binding protein Homo sapiens 65 29 49 60 Pe A M pann ta BioGrid Selecting BioGrid interactions not found here opens a new window with the details of the missed interactions as shown below Note that the overlap is performed after data filtering is applied thus care should be taken when analyzing
61. id 419 109809739 51574 LARP 955 20 109809741 66 860 Laribonucleoprotein domain family member 7 Homo sapiens 53 14 25 80 oy E M panita In this example we will filter the data shown above by applying the following filters 1 click the background button and select the FLAG_top_contaminants list from the dropdown menu The background lists are user defined and controlled via the Manage Lists and Protocols option 2 proteins detected with a Mascot score lt 60 will be removed 3 proteins with lt 20 sequence coverage will be removed 4 proteins detected with a single unique peptide will be removed 5 proteins detected in gt 25 of the samples in this project will be removed After applying filters the list of hits is reduced see the disappearance of C1QBP NCL and NPM1 which are common contaminants while SART3 LARP7 and LSM8 remain The filters can be modified and sorting repeated ProHits does not remove any data from the dataset but only displays filtered lists Hide Filters Experiment Filters V Score lt 60 O Expet gt 1 gt M Coverage lt 20 Vi Peptide Unique Peptide lt 2 4 V Frequency gt 25 m V FLAG_top_contaminant L H 1 1 V Carry Over E V Spill Over O Auto MW Exclusion Bio Filters O Ribosomal ii C Cytoskeleton Wi O Bait ml Y Keratin 4 C Artifact Protein F C Translation Elongation Factor E DEAD H Box C Albumin W E A Biocria ov
62. io Filters Select desired C Ribosomal C Cytoskeleton Bait V Keratin parameters _ Artifact Protein C Translation Elongation Factor DEAD H Box _ Albumin BioGrid overlap C Physical HTP W Press Go to CJ Phys C Genetic NON HTP gt Select to highlight the MH 64 113 177 255 347 454 574 BioGrid overlap if desired To apply filters press apply the filters 709 Total Peptide Number Update Frequency GO BioGrid overlap Physical HTP Y Go W Physical NON HTP O Genetic HTP Y7 C Genetic NON HTP A This generates a modified list similar to the process o described in the Bait report section If selected the overlap with BioGrid is indicated by stars or triangles in the list below 28 64 113 177 255 347 454 12 WASL_pelletACN Flags Merged croup 1 Note that mousing over any of the entries shown below will pop up a menu box listing the scoring details Select to overlap with data Missed interactions with selected filters are indicated 574 74 look literature curated 9 Total Peptide Number LletBCN Flas 11 EF4A2_pelletO N Flag 10 EIFd4A2_pelletC N Flag 4 3 WAaSL_ne 14 RAF1_pelletACN Flag 15 RAF1_pelletBCN Flag at Options to visualize the data in Cytoscape or export the comparison view Note that the filtered results will be exported DOG BioGrid PF3 Bio
63. its Project Demo Human Gel Free Submit Gel Free Sample Add New Bait Bait List Column Display Set Experiment status color keys Bait groups select to compa Total Baits 5 1 Page 1 Gemtiane gi 10 FLAG alone N Filag Prohits Administrator E gt N Flag 4506401 Prohits Administrator a im i RAF1_pelletA a Interaction detection method MI 0007 anti tag coimmunoprecipitation Call type 293 Fip In T REx Invitrogen Ij Tet Inducible Fip in 293 2010 1 1 J Human cell lysis and FLAG IP GC protocol 2010 1 2 9 Simple in solution trypsin digest of AP samples 2010 1 3 gt Jj Pressure bomb oad LC MS on LTQ BL protocol 2010 1 4 Expt Notes List here additional notes e g 1 changes to standard protocol above 2 other experimental details not captured above 3 cross references to internal databases 4 cross references to notebooks 5 data ownership notes N Flag 51702526 Prohits Administrator a N Flag 83700235 Prohits Administrator N Flag 47271406 Prohits Administrator Total Baits 5 1 Page 1 Once your baits and experiments are entered you can create one or multiple samples to be linked to the bait and experiment The number of samples you create for a given experiment depends on your experimental set up We tend to use different samples from a single experiment to represent technical replicates i e different MS runs from the same biological sample where all conditions are t
64. lag EIF4A2 1974 83700235 1720 363 25 64 6 60 20 4 14 RAF1_pelletA N Flag RAF1 5894 4506401 1620 353 20 50 9 20 20 5 13 WASL_pelletB N Flag WASL 8976 51702526 1320 303 21 60 4 20 20 6 15 RAF1_pelletB N Flag RAFL 5894 4506401 1560 258 20 53 9 20 20 a 12 WASL_pelletA N Flag WASL 8976 51702526 1457 244 23 57 2 20 20 8 9 MEPCE_pelletB N Flag SART3 9733 7661952 1683 242 24 35 8 20 20 a a 9 MEPCE_pelletB N Flag MEPCE 56257 47271406 1480 184 24 53 7 20 20 10 9 MEPCE_pelletB N Flaqg LARP 51574 109809739 1121 176 19 33 5 20 20 113 14 RAF1_pelletA N Flag YWHAG 7532 21464101 321 170 3 27 1 20 20 12 14 RAF1_pelletA N Flag YWHAQ 10971 5803227 455 168 4 26 5 20 20 akg 14 RAF1_pelletA N Flag YWHAH 7533 4507951 295 166 3 22 8 20 20 14 8 MEPCE_pelletA N Flaqg MEPCE 56257 47271406 1106 152 19 43 8 20 20 15 15 RAF1_pelletB N Flag YWHAH 7533 4507951 472 107 4 33 3 20 20 16 8 MEPCE_pelletA N Flaqg SART3 9733 7661952 1463 105 20 26 9 20 20 17 10 EIF4A2_pelletC N Flag EIF4G3 8672 10092601 1598 76 25 23 3 20 10 Export matrix provides a view similar to that displayed in the Comparison page with the option to export only the parameter currently displayed e g spectral counts or the option to list all parameters inside each cell Again a csv file that can be opened and modified in Excel will be created View only the displayed value here total peptide counts A B F G H feel Gonereted date eT Project Name Demo Human Gel F
65. lters Click on the Show Filters button within the results page to display the administrator defined Bio and Experimental filters see admin office for details of the filtering options and background lists see Manage Protocols and Lists that can be applied to the data in this project On the left is the filter list and the graph on the right indicates the number of proteins that would be removed by activating each of the filters Filters are activated or de activated by clicking their associated checkbox Once the desired filters are selected press Apply exclusion to remove associated proteins from the search results list Note that the default frequency filter is set in the admin office module when creating the project and that this value is listed when you select the project from the home page see page 3 In the case of the Demo Human Gel Free project shown here the frequency filter was set at 3 meaning that a protein detected in gt 3 of samples within the project is flagged as shown by the dark green icon in the results table You do not need to use the default filter and can modify this frequency cut off as needed Also note that the frequency is not automatically recalculated every time you add a search result to ProHits to recalculate the frequency use the Update Frequency button on any Report page Hide Filters Experiment Filters 1 Select desired filters V Score lt 60 O Expect gt
66. lues by typing their description and pressing Add Values that are not yet linked to an entry are followed by a red X Pressing X deletes the entry Note that for this category we have used PSI MI 2 5 terms to facilitate later deposition in interaction databases Controlled Vocabularies Experimental Details Click to add new selection or option Edit selection Interaction detection method MI 0007 anti tag coimmunoprecipitation x MI 0006 anti bait coimmunoprecipitation MI 0096 pull down gt To define new categories press the button next to Edit selection This allows you to enter a new category gt To import a category from another project to which you have access simply click the checkbox associated to the category under the Edit selection option to transfer the category and associated values to current project Controlled Vocabularies Experimental Details Click to add new selection or option Edit selection Add new selection Interaction detection method x Import selections from other objects Selection Project Cell type Demo Yeast Gel Free Tissue source Demo Yeast Gel Free Controlled Vocabularies Experimental Details Click to add new selection or option Edit selection Add new selection Interaction detection method Cell type Tissue source Import selections from other objects Selection
67. mapping your terms to PSI MI 2 5 whenever possible Epitope Tags Name OLSID add new OLS Lookup Location MI 0340 n terminal position OLS Looku Description
68. matted and size 17x17 pixels image Click here to download a photoshop template icon Experiment Groups add new import from other projects Sample Groups add new import from other projects Export Versions add new import from other projects Export version ProHits allows you to flag a group of samples e g for inclusion in a publication or export to a third party gt To create an Export Version press add new This will open a new menu with the default abbreviation Versionl VS1 and Icon a yellow star with the number 1 Subsequent versions will automatically be numbered VS2 VS3 etc and the number inside the star will similarly increase We suggest that you provide a meaningful short name and an accurate description of each Export Version Export Versions add new import from other projects Name Type Export Description Abbreviation VS1 Icon tr Epitope Tag Lists Select the Epitope Tag Lists entry from the Manage Protocols and Lists This lists all tags available to the local ProHits projects Clicking on the sign expands the details of the epitope tag We have mapped the current epitope tags in the demo database to PSI MI 2 5 using the Ontology Lookup Service OLS at the EBI A link page is provided that allow retrieval of additional information Epitope Tags add new ae iiae DF C Flag OLSID MI 0518 OLS Looku Location
69. n before transferring the selected files PA Sample List Samples Selected Samples Merge groups BaitID GeneName Tag SamplelID SampleName BaitID GeneName Tag SamplelD SampleName new group color pay 6 MEPCE N Flag 9 MEPCE_pelletB 6 MEPCE N Flag 8 MEPCE_pelletA Control 8 WASL 12 WASL_pelletA O Unmerge 8 WASL 13 WASL_pelletB 9 RAF1 14 RAF1_pelletA 9 RAFl 15 RAF1_pelletB 7 EIF4A2 N Flag 11 EIF4A2_pelletD 7 EIF4A2 N Flag 10 EIF4A2_pelletC D You can group any set of additional files by first clicking on the multicolour icon to select a new group then transferring the given files to the right side The listing order will be as follows The control group will be listed first followed by all other groups in the order selected by the user followed by all individual entries in the order selected by the user Note that within the same group hits will be combined and only the maximal value for each of the properties will be reported 1 To combine multiple Baits into one group click first on the multicolour icon and select desired colour J Sample List z z 6 Samples Selected Samples Merge gro p BaitID GeneName Tag SamplelD SampleName BaitlD GeneName Tag SamplelD SampleName new group col 6 MEPCE N Flag 9 MEPCE_pelletB 6 MEPCE N Flag 8 MEPCE_pelletA WASL 12 WASL _pelletA WASL 13 WASL pelletB RAF1 14 RAF1_pelletA RAF1 15 RAF1_pelletB EIF4A2 N Flag 11 EIF4A2_pelletD EIF4A2 N Flag 10 EIF
70. n datasets are user defined They may consist for example of proteins that adhere to resin in the absence of a bait or of any other proteins that the user wishes to exclude One project may be linked to multiple non specific data sets A single non specific data set can be selected to filter hits both in the Bait or Sample Report and in the Comparison pages of the Analyst module Only the Admin has permission to modify or add a non specific background set Project Demo Human Gel Free Species Homo sapiens human Modified by Modified date Add New Close Window Background Set gt FF g Import from other projects Close Window Note that an efficient method to generate a non specific filter set utilizes the ProHits comparison tool First select multiple control runs and merge them into a single Control group This will open up a Comparison page with a single column called Control Group displayed in yellow As before the maximal value for the parameter visualized is displayed e g spectral count Apply filters e g number of unique peptides protein coverage etc desired and select Export table to export a comma delimited file csv Save this file on your hard drive and go to any Bait report page Note that any Excel or text file that lists the NCBI Gene ID may also be used Browse the file to be uploaded select delimiter and press upload file Select the add as new
71. nts and may be useful for spi Hurov K E Luo J Bakal immunotherapy This gene product is found to be an imp Lerenthal Y Shiloh Y factor for HIV 1 gene expression and viral replication It gt SMAX ere ust oa TITLE ATM and ATR pig hg en anal transiently with U6 and U4 U6 snRNPs during the recyclir JIC art responsive to amage JOURNAL Science 316 5828 1160 1 spliceosome cycle This encoded protein is thought to be smat nuclear nbonucieoprotein polypeptides B and 51 a oat Bema reraoon toes PUBMED 17525332 regulation of mRNA splicing provided by RefSeq gt ee Gene Ontology View List PRP4 pre mRNA processing actor 4 homolog yeast Gene Oratogy view List e Genomic regions transcripts and products G I PRPS pro MANA processing tactor 3 homciag S cerevisiae em minus strand Go to reference sequence details Tyour err ces SMT homolog U6 smali nuciear RAIA associated S carevisian Gone onsoiogy vw ust a a OO O S NC_000012 11 a BigGrid Option details Pressing the following icons in the Option column will retrieve the peptide list from the search engine for each hit or the entire search results file MarR IS gENCES Hit Information 7661952 Download DAT Instrument ESI TRAP Ser elEXpect 14638 MATRIX rene ar Satan ice Mascot Search Results Email gliu mshri on ca Search title demo human MS data file 8_MEPCE pelletA mgf Database Human_RefseqV33 RefseqV33_200
72. one gt Click the green arrow to transfer the protocol from the source project to the destination project modify if needed and press Save You can export protocols linked to a project to a CSV file that can be opened in Excel or similar programs The Detail column contains the full text of the protocol Charts SmartA FO G H Creation Da Creater 1 7 10 Prohits Administrator 1 7 10 Prohits Administrator 1 7 10 Prohits Administrator 1 7 10 Prohits Administrator oneets gt A B c D E ID Name Type Project Name Detail 21 Trypsin digestion on magnetic beads Peptide Preparation Demo Human Gel Free Proteins immobilize 17 In solution digest of IP samples Peptide Preparation Demo Human Gel Free Trypsin 1 ug Sigmz 19 Growth and gal induction for HTP yeast project Biological Material Demo Human Gel Free Plasmids encoding 15 Tet Inducible Fip In 293 clone Biological Material Demo Human Gel Free Human taxid 9606 Experimental Editor Select the Experimental Editor entry from the Manage Protocols and Lists You will see a list of the categories already defined for your project Controlled Vocabularies experimental Details Click to add new selection or option Edit selection Interaction detection method gt To view the values already entered under the interaction detection method category click on the button to expand this category You can add additional va
73. one B09_38 Prohits Administrator 6 P1G1 _IP47 G546 jpg 55 000 3 None c02_34 Prohits Administrator 2 P1G1_1P47 G546 jpa c02 32 000 3 None c04_1 Prohits Administrator 1 P1G1_IP47 G546 jpa c04 25 000 3 None January 12 2010 10 23 am When setting up the acquisition on the mass spectrometer the folder name here 20080715_YDP00155_A1_P1 as well as the Raw file names e g BO6_35 need to match these above The Comparison tool ProHits has a built in comparison tool that allows you to look at the results of several experiments side by side You can perform comparisons at the bait level or at the sample level and compare the results from the search engines e g Mascot or X Tandem or the TPP For this demonstration we will perform a comparison at the sample level using the Mascot search engine Comparison Project Demo Human Gel Free instructions To use the Comparis Multiple samples ma In a bait comparison peptides identified fo Data may be merged Baits Samples windo When all Baits Sampresemororgroups aretugpeu anc Teauy Tor compansyr p venerate Rep ONT e There are two types of color codes in these ults page The hit property cgfor code indicates hit property vakfes by cofor gradient For example the default hit property is total peptides blue the darker the sige the higher the number of peptig s observed The shared hits gefor code is ised to indicate hits in common between sam
74. ou must first specify a project create an entry for the protein of interest bait and define experimental conditions Typical gel free samples are eluates from an affinity purification Enter a bait gene name species tag Specify experimental conditions cell growth lysis IP MS etc Add samples Samples will be linked to the MS data management module Sample entry for gel based projects is similar with the exception that a gel is specified prior to the selection of a bait Typically samples are gel bands and all bands from the same lane are entered under the same Experiment project Enter a bait gene name species tag bait bait Access to projects Projects are created by your administrator in the Admin Office ProHits module and access is granted to users Projects can be specific to a research group or an individual to a given organism or specific methodology etc The creation of a new project is defined in the Admin Office manual Specify experimental conditions cell growth lysis IP MS etc Add samples Samples will be linked to the MS data management module When you log into ProHits with your user name you can see the list of all of the projects that you have access to You may have different privileges for each project gt Highlight the desired project then hit Select Fa ProHits Analyst Protein High throughput Solution
75. perimental details section will by default prompt you to define a lane on the gel and guide you through the entry of individual band samples in the autosampler plate that you will use for data acquisition Simply clicking on a plate well will create an associated sample you can add the intensity of each band on the stained gel as well as the approximate molecular weight Delete al Next Level Modify Band Sample Project Demo Yeast Gel Submit Gel Sample Upload next Lane Back to Lane Back to Experiment eat Gene ID 855143 Gel Name IP47 G546 LocusTag GeneName YMR116C ASC1 Gel Image P1G1_1P47 G546 jpg Bait MW 34 810 kDa Method of Staining Coomassie Clone Number YST1207 Gel Type 1 D Exp Name IP47 Uploaded 2008 07 15 14 12 53 Created by Prohits Administrator 2008 07 15 Uploaded by Prohits Administrator O Lane Code ASC1 IP47 G546 Lane Number 3 Lane Notes YDP00155 Plate Layout Prohits Administrator 2008 07 15 15 24 17 Prohits Administrator 10 11 12 2008 09 19 16 05 14 2008 09 19 16 05 14 2008 09 19 I lt ES ES Eal New Plate a OT 1 35 B06 BEE 100 000 kDa None PE 2 34 co2 Sa 32 000 kDa Noe BDT ii F BEHET soo ve MIE E 4 B07 6 0 O O Cancheck 75 kDa None B Continue entering all desired bands from the selected lane or use the navigation options at the top of the page to upload samples from the next lane return to the list of all lanes or return to the experimental
76. ple result pages 4 Manage Protocols and Lists allows you to create and maintain experimental protocols controlled vocabularies background lists group lists and epitope tag lists Access to these pages is defined by the ProHits Administrator 5 Other Tools provides additional functionality Co IP Report allows you to input results from follow up experiments aimed at confirming interaction pairs by immunoprecipitation immunoblotting Export Functions allows you to export filtered or unfiltered lists of mass spectrometry results Note that export functions are also available within each of the Individual Report or Comparison pages 6 Advanced search allows you to query your project for genes keywords and or controlled vocabularies Creating samples and viewing individual reports To learn more about the different functions of ProHits Analyst we will navigate through the Analyst module by creating new baits and linking them to entries from the Data management system We will then explore the functions available in the Analyst module We will go through the process of adding a gel free sample and explore the results for this type of project We will then briefly review the differences between submitting gel free and gel based samples Adding a Gel Free sample To create a new sample to be linked to a search result file you will first specify a bait then an experiment and then a sample To submit a sample you have two options 1 cre
77. ples ected Baits the same b lotein hit are 2 The unselected baits are listed on the left side Use the Sort by function to reorganize 1 Select whether you want to compare Baits or Samples and TPP or results from the search engines er of 5 Click to s F T Sample List o Se Samples Sel cted Samples Merge groups BaitID GeneName Tag SamplelID SampleName BaitID GeneNanfe Tag SamplelD SampleName new group color gay 10 FLAG_alone N Flag 17 FLAG_alone_pelletD 10 FLAG_alone N Flag 16 FLAG_alone_pelletC Q Control 9 RAF1 N Flag 15 RAF1_pelletB Unmerge RAF1 N Flag 14 RAFl_pelletA WASL N Flag 13 WASL_pelletB WASL N Flag 12 WASL_pelletA EIF4A2 N Flag 11 EIF4A2_pelletD EIF4A2 N Flag 10 EIF4A2_pelletC MEPCE N Flag 9 MEPCE_pelletB MEPCE N Flag 8 MEPCE_pelletA t ann nN OW C gt gt f lt Hit property color code Sort by group type Bait O Experiment O Jample Shared hits color code O Sort by group 3 Remove l Unused colors Then sort by _ Bait ID Sort sample list Select the desired baits to be compared You can sort by Bait ID Gene name Protein ID or by any of the user defined flags that were used for the project Press the gt gt arrow button to transfer the baits to the Selected Baits window You can transfer files one at the time or by large groups The files are added to the list in the order selected This will also b
78. ransferred Note that if there is more than one entry mapped to a given gene the user can select which one is to be parsed into ProHits ProHits also allows you to indicate which epitope tag you are using by selecting from options in the Epitope Tag menu you can also add new tags using the Manage Protocols and Lists option If the sequence of the bait is mutated relative to the HUGO sequence you can also enter this in the Bait mutation box Delete esa Next Level Modify gt Next th Bait Report D Bait Notes Baits Project Demo Human Gel Free Submit Gel Free Sample Add New Bait Bait List New gene for IP experiment No gene control or non IP experiment rs Bait ID Species Homo sapiens human 4 Gene Name MEpcE pitope Tag N Flag Detail Bait Mutation If the bait sequenc angst enter the wild type gene name to gef its protein information Tye bodifu the eno Nama as Wo wich and writs the madifieatian datai in Description field A A p Specify species official gene symbol Epitope GenelD tag and Bait mutation if applicable LocusTag This field is ignored if Gene ID is specified when you click Get Protein Info button ProteinID 3 ce i z en Click get protein info a Femy Gene ID 56257 _I7I7 Gene Name MEPCE persist inde ia oe aes Description Species Home sapiens human Description methylphosphate capping ery Check information for accuracy
79. re additional notes e g 1 changes to standard protocol above 2 other experimental details not captured above 3 cross references to internal databases 4 cross references to notebooks 5 data ownership notes You are now ready to explore your results Use the left hand side of the ProHits Analyst main page to view Report by Bait and Report by Sample The interface for the Bait and Sample reports is very similar Here we provide an example for the Sample Report Bait versus Sample view For some projects you may have a one to one correspondence between bait and sample For other projects you will have multiple samples linked to the same bait Opening the Bait Report when two or more samples are linked to the bait will generate sequential protein hit lists for each of the samples linked to the bait ProHits does not recalculate scores or peptide numbers but indicates in bold proteins detected in more than one sample mousing over bolded names activates a pop up window that provides details about the samples and hit scores If you wish to explore each sample individually use the Report by Sample link instead Navigating through the results Now that we have entered baits linked and parsed search results it is time to look at search results In this example we will start from the Report by Sample page for MEPCE_pelletA 2 The following page appears displaying the results from your search engine th
80. ree 2 PID SC PT PU C F SF Peptide ID Score Total Peptide Number Unique Peptide Numbe Cover 3 4 17 FLAG_alone_pelletD 8 MEPCE_pelletA N Fl 9 MEPCE_pelletB N Flac 1 5 6 Gene ID Gene Name Total Peptide Number Total Peptide Number Total Peptide Number 1 _7 194018537 18 8 195539395 6 9 10213 PSMD14 a 10 56257 MEPCE 152 184 11 9733 SART3 105 242 ah 51574 LARP 53 176 fab 51691 LSM8 49 35 14 217272892 26 30 phy 9129 PRPF3 26 32 16 221136939 25 40 pol 25804 LSM4 17 11 18 1025 CDK9 16 FEN 2021 ENDOG 15 34 20 10614 HEXIM1 12 21 57819 LSM2 8 9 View all parameters gt A B D E F G H 1 Generated date 2010 January 15 Project Name Demo Human Gel Free 2 PID SC PT PU C F SF Peptide ID Score Total Peptide Number Unique Peptide Numbe Coverage Frequency Sub Frequency Zoom of the details inside each cell PID SC PT PU C F SF 56790935 500 16 10 20 70 11 88 75 Legend PID Protein ID NCBI Entrez Protein SC Mascot Score PT Total number of peptides PU Number of Unique peptides C Percentage of the protein sequenced F Frequency of occurrence of the protein in the entire dataset SF Frequency of occurrence of the protein amongst compared baits samples 3 4 16 FLAG_alone_pelletC N Flaq 17 FLAG_alone_pelletD N Flag 8 MEPCE_pelletA N Flaqg 9 MEPCE_pelletB N Flaq 5 6 Gene ID Gene Name Protein ID Total Peptide Number Total Peptide Number Total Peptide Number Total Peptide Number 7 194018537
81. ross the bait purifications Change sorting options Select to expand filter options Color code Hit property color code Sortby Total Peptide Number Sample ID Control Group Descending Ascending Click to apply filters 0 7 28 64 113 177 255 347 454 574 709 Total Peptide Number Update Frequency Rows list individual hits Click on gene name to go to NCBI gene BioGrid to view Cytoscape Export tab Columns list baits b bi bi bi n n interactions for this protein 6 u u u ii or groups of baits al Z l Protein ID to open the NCBI 2 5 2 Hits Protein page and Peptide z 2 i Comparison to view peptide ja 2 2 2 2 ol ol g 4 a S T details g a A a T on 3 3 rad Lu W TER ee g y a 3 z 9 Gene Nag Protein e eaeic PRMTAR BioGrid 20070220 coding display the WDR77 BioGrid 13129110 KIF 11 BioGrid 13699824 selected property FLNA BioGrid 116063573 here total KRT1 BioGrid 119395750 peptides Mousing IVNS1ABP BioGrid 24475847 ays HSPA1B BioGrid 167466173 over a hit will pop TUBB BioGrid 29788785 up another box TUBB2C BioGrid 5174735 i LOC651751 BioGrid 169218253 with hit details PRPS2 BioGrid 4506129 PRPSAP2 BioGrid 4506133 TUBB2A BioGrid 4507729 CLNS1A BioGrid 4502891 CCT8 BioGrid 48762932 NMM A Me M afrnrant The default display is with Total Peptide Numbers spectral counts and the default sor
82. t module and lists the search tasks bearing the selected keywords complement component 1 g 100 subcomponent binding protein Searching Bait Protein Description You can search for a keyword inside the Description field e g squamous in the example above by allowing wildcards on both sides In other words the entire field is captured not individual words and any partial field e g squamous or carcinoma must be preceded and or followed by wildcards Note again that such searches may be very slow Searching in Experimental Details controlled vocabularies The search function also allows you to search or limit your searches based on selected controlled vocabulary Simply press Select bottom right corner of the Experimental Detail section This will take you to the Experimental Details controlled vocabulary section where you can select categories values to be passed to the Advanced search page Restricting searches by date You can restrict search results by date Simply press the select button in the Date field to open a drop menu Using logical operations You can combine several keywords simply separate them by spaces to search for at least one of the words all words in any order or the exact phrase within a field such as Description Note that the all words and exact phrase operations only apply within a field Alternatively you can use the at l
83. t the user wishes to exclude One project may be linked to multiple non specific data sets A single non specific data set can be selected to filter hits both in the Bait or Sample Report and in the Comparison pages of the Analyst module Only the Admin has permission to modify or add a non specific background set Project Demo Human Gel Free Species Homo sapiens human Modified by Prohits Administrator Modified date 2010 01 09 Add New Close Window Background Set FLAG_top_contaminant Import from other projects TAT SY ACTA ASMA CFTD CFTD1 G CFTDM ene 58 ACTA1 eI MPFED BioGrid fs NEM1 NEM2 NEM3 RP5 1068B5 2 ACT Gene 345651 ACTBL2 DKFZp686D0972 BioGrid ATP5A ATP5AL2 ATPM 498 ATP5A1 MOM2 Gene OMR BioGrid ORM hATP1 ATPMB 506 ATPSB ATPSB Gene MGC5231 BioGrid afa a as To manually add a protein to a background list press Add New You will then be prompted to enter a new contaminating background protein You can simply enter a gene name and species and press Get Protein Info Press Add to include this protein on the background list GenelD 58 Get Protein Info RP5 1068B5 2 This field is ignored if a Gene ID is specified when you click Get Protein Info ACTA1 Gene Name This field is ignored if a Gene ID or a Locus Tag is specified when you click Get Protein Info Species Homo sapiens human r LocusTag Set Name If you do not specif
84. t that is automatically assigned by ProHits The Gene Name and Tag are indicated and the Protein ID is the accession number from the selected database e g NCBI GI The User column is automatically assigned to the user who created the sample i e the person who has signed up in ProHits Note that on many of the ProHits pages you will find standard icons as seen at the top of the Bait Report page Delete Fat Next Level Lal Modify gt Next th Bait Report oe Ea Bait Notes 1 To remove unused material press the delete icon The delete function may be used to remove baits experiments or samples but only if no information has been entered If you wish to delete a bait experiment or sample for which information has been entered start by deleting the information at a lower level and work your way up Note that there is an Admin control for the permissions to insert modify and delete entries and you can only delete your own entries 2 The next level tree icon allows you to navigate down in the data structure i e from bait to experiment to sample 3 The Modify icon allows you to change the information you entered for a bait experiment or sample 4 The green arrow Next icon allows you to submit information and or exit a page after data has been entered 5 The Bait report graph icon shows you the mass spectrometry results for the selected bait We will review this in detail later 6 Finally the
85. tails not captured above 3 cross references to internal databases 4 cross references to notebooks 5 data ownership notes Click the Data Management tab from any page of the Analyst module parse the hits as described in the Data Management section and return to the Analyst module Once hits are parsed either from the Data Management or the Analyst module a new purple coloured tab will appear in the status bar in either Bait Report or Sample Report pages indicating the total number of hits identified sum of hits if more than one search engine was used In the Options column a new graph icon appears clicking this link brings up the search results for each sample Here we are showing MEPCE_pellet A in the sample report view Samples Project Demo Human Gel Free Column Display Set Experiment status color keys Sample groups select to compare Total Bands 10 1 Page 1 Exp Status Cntio Show groups Bait WeExperiment CsSample W Version aaie 6 MEPCE Prohits Administrator 9 2010 01 05 Interaction detection method MI 0007 anti tag coimmunoprecipitation Cell type 293 Fip in T REx Invitrogen fj Tet Inducible Fip In 293 clone 2010 1 1 J 293 cell lysis and FLAG IP 2010 1 7 In solution digest of IP samples 2010 1 7 p MEPCE_pelletA a Ge Pe J Pressure bomb load on LTQ 2010 1 7 J 8_MEPCE_pelletA RAW 167 540KB detail Bf of Hits 590 TPP hits 779 Expt Notes List he
86. ting option is by descending number of spectra starting by the left most bait or group Note that these sorting options can be modified In particular ProHits recalculates and sorts using the following parameters PPPRP Total Peptide Number 7 28 64 113 177 255 347 454 574 709 TN Unique Peptide Number 5 14 18 23 28 32 37 41 4 9 6 tz SME 0 9 17 26 34 43 51 60 68 77 85 Project Frequency 0 10 20 30 40 50 60 70 80 90 100 Mascot Score 0 23 92 208 370 578 832 1132147818712310 0 2 9 20 35 55 79 108 141 179 221 GPM Expect TPP Probability O 0 010 040 090 160 250 360 490 640 81 1 In addition to the sorting options ProHits Comparison allows you to filter your data in a manner similar to the filtering options in the Bait Report page gt To access the filtering option select Click to apply filters Sort by An expanded menu allows Click to remove filters Color code Hit property color code Total Peptide Number Sample ID Control Sample Comparison Shared hits color code Experiment Filters Mascot Score lt Peptide you to select criteria for removal of proteins from the Comparison list 60 Unique Peptide es overage lt 20 2 1 Frequency Project Frequency bad E 25 Select desired filtering options vV Background Set FLAG_top_contaminant La C Carry Over C Spill Over Auto MW Exclusion B
87. ule only those files not previously linked to another entry will be displayed To modify an existing link you need to go back to the Data Management module remove the link to the initial file so that it can be made available to link to an entry either through the Analyst or Data Management modules Link Prohits sample to raw file Project Name 2 Demo Yeast Gel Free Bait 19 ALK2 Experiment 29 ALK2_sampleA Gel Gel free Sample 32 ALK2_sampleA Sample information Project 2 Demo Yeast Gel Free Machine Name LTQ DEMO Get raw file info t LTQ_DEMO Raw file Project Demo Yeast Gel Free Total Folder 1 1 Page 1 S LTQ_ DEMO 3 YST_GelFree_P2 O 3461_TPK2_HA RAW 75 834 KB 20100108 Do 3459_TPK2_HA RAW 81 859 KB 2010 01 08 D 03495_SWE1 RAW 94 364 KB 2010 01408 D 03493_SWE1 RAW 93 520 KB 2010 01208 D 03427_TPK2 RAW 87 002 KB 2010001408 Do 3423_TPK2 RAW 84 650 KB 2010 01 08 D 03367_Swe1_b RAW 84 383 KB 2010 01 08 D 03366_Swe1_B RAW 86 073 KB 2010 01 08 D 03354_MPS1_b RAW ss i96 KEy 2010 01 08 Do 3353_MPS1_b RAW 72 870 KB 2010 01 08 D 03325_Swe1_HA_b RAW 82 400 KB 2010 01 08 D 03324_Swe1_HA_b RAW 87 740 KB 2010 01208 O 03312_MPS1_HA_b RAW 87 665 KB 2010 01 08 O 03311_MPS1_HA_b RAW 87 448 KB 2010 01 08 D 03142_ALK2 RAW 90 074 KBy 2010 01 08 3141_ALK2 RAW 69 217 KB 2010 01 08 0 2810_ALK2_HA RAW 96 558 KB 2010 01 08 D 2809_ALK2_HA RAW 3 0s6 KB 20100120
88. wed and a new protocol that will be assigned a different protocol number will need to be created Pro tocols Project Demo Human Gel Free Export protocols Biological Material add new import from other projects Tet Inducible Fip In 293 Protocol ID 10 Protocol Type Biological Material Project Demo Human Gel Free Protocol Name Tet Inducible Fip In 293 Created by Prohits Administrator Creation date 2010 01 05 Protocol Detail Human taxid 9606 cells Fip In T REx 293 cells passage 15 from S Angers laboratory were transfected in a 6 well format with 0 2 ug of tagged DNA pcDNA5 FLAG protein OpenFreezer V4071 and 2 ug pOG44 OpenFreezer V4134 using lipofectamine PLUS Invitrogen according to the manufacturer s instructions On day 2 cells were trypsinized and passaged into 3 x 10 cm plates in a two fold dilution series On day 3 the medium was replaced by DMEM 5 Fetal bovine serum 5 calf serum 100 units ml pen strep 200 pg ml hygromycin Medium was replaced every 3 4 days until non transfected cells die and isolated clones are 2 mm in diameter 13 15 days The clone position was marked at the bottom of the plates Stable cell clones were picked by trypsinization using 2 mm sterile 3MM filter papers dipped into trypsin Paper circles are transferred into 24 well plates each well containing 1 mi of complete growth containing 10 Tet system tested FBS Clontech 631106 medium with hygromycin Clones were
89. y a pre entered non specific set ProHits will allow you to create a new one press Confirm after entering the non specific set name To import a contaminant list from a different project press Import from other projects and navigate through the menus Ni on spec ific bac kground import from other project Projects Demo Human Gel Free 4 Non specific Set f FLAG_top_contaminant Set Name Add as new O Append to existing Select a group oeeo cantons create e ACTA ASMA CFTD CFTD1 58 ACTA CFTDM Gene MPFD BioGrid NEM1 NEM2 NEM3 RP5 1068B5 2 ACT Gene T 345651 ACTBL2 DKFZp686D0972 BioGrid You now have your own background set that can be used for filtering both in the bait sample report pages and in comparison We recommend using caution when creating these sets some proteins that are true interacting partners for a given bait may also be present usually in lower amounts on the background list It may be a good idea to only include on this non specific background list proteins detected across more than one control run with a high number of peptides Group Lists ProHits allows the definition of new groups for any given project As described earlier groups are added to baits samples by selecting the Notes Option Groups act like flags and are displayed in the status bar in the Report by Bait or Report by Sample pages These groups can help

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