SIEVE 1.1.0 User Guide Version B
Contents
1. Understanding the ChromAlign Process When you send an experiment for analysis after selecting sample and control files and setting parameters SIEVE performs an automatic aligning process called ChromAlign This process aligns the chromatograms of the sample files with the first control file correcting chromatographic shifts The following factors can contribute to chromatographic shifts which are part of the challenge in biomarker discovery driven by differential analysis The difficulty in reproducing LC MS experiments Aging of separation columns changes in sample buffer or content or fluctuations in temperature and sample flow The absence of any prediction model that explains peptide elution in chromatographic columns and allows alignment of eluting peaks through the accurate modeling of peptide elution The following sections provide detailed information about the ChromAlign process Outlining the ChromAlign Process Starting the ChromAlign Process ChromAlign First Level Alignment ChromAlign Second Level Alignment Aligning Over Different Abundance Levels Aligning Over Chromalign Time Shifts ChromAlign US Patent applied for Thermo Scientific SIEVE User Guide 27 A Understanding the ChromAlign Process Outlining the ChromAlign Process Outlining the ChromAlign Process Start with Original Chromatograms After selecting samples and determining the control data set send the A and B control and sample2. 2007 Thermo Fisher Scientific Inc All rights reserved i wy PM SONE Microsoft Windows XP is a registered trademark of Microsoft Corporation in the United States and other countries SIEVE ChromAlign LTQ XL LTQ Orbitrap LTQ FT Ultra and BioWorks are trademarks and Sams is a registered trademark of Thermo Fisher Scientific Inc DecisionSite is a trademark and SRS is a registered trademark of Spotfire Inc SEQUEST is a registered trademark of the University of Washington Thermo Fisher Scientific Inc provides this document to its customers with a product purchase to use in the product operation This document is copyright protected and any reproduction of the whole or any part of this document is strictly prohibited except with the written authorization of Thermo Fisher Scientific Inc The contents of this document are subject to change without notice All technical information in this document is for reference purposes only System configurations and specifications in this document supersede all previous information received by the purchaser Thermo Fisher Scientific Inc makes no representations that this document is complete accurate or error free and assumes no responsibility and will not be liable for any errors omissions damage or loss that might result from any use of this document even if the information in the document is followed properly This document is not part of any sales con
3. Make changes to the parameters as needed Click Run 2 Setting up a Procedure Identifying Proteins and Peptides If you selected Show advanced progress info in the Submit Analysis view a DOS window displays the progress of the procedure At the end of the SEQUEST database search SIEVE opens the Spotfire application 3 To see a consolidated summary of SIEVE results click the Table View tab in the Spotfire main view This table includes columns displaying the pValue and intensity ratio for each frame as well as the SEQUEST search results if applicable SIEVE provides the results of the differential analysis in a table view so that you can check the protein or peptide columns and select a frame with useful protein or peptide information For information about the various Spotfire views shown as tabs at the bottom of the screen or the sliding scale parameter settings see Appendix C Using Spotfire to View Analysis Results Figure 12 Table view in Spotfire post SEQUEST search Vig SIARCH sfs Spotlire DecisionSite z 39d 4 a amp a6r NB H303 t 234 lola M TFSEAEFPFNPEI M TFSEADFPENPSI KLVNANGEAVYCK K DFNVGGYIQNAL 2485 013 28013 328E 013 36X 013 ENE orto 258x012 204E011 204E411 VEO 2 001 652001 SME 356E 011 164E 010 165E410 165 010 26010 282 0 0 314E010 45E010 Tw 745 010 SOE 0 0 124E 008 136E008 129 08 13x 008 242 008 268 008 IIFA Thermo Scientif
4. 1439 records visible 100 00 0 marked Thermo Scientific SIEVE User Guide 43 C Using Spotfire to View Analysis Results Spotfire Views Results Summary Table SIEVE presents the results from an entire biomarker discovery experiment in an easy to interpret interactive table Sort data by ratio of change pValue protein identification m z value or XCorr for example with the additional flexibility of modifying columns to suit individual experiments Figure 29 Spotfire Results Summary table Viga SLARCH sfs Spotfire DecisionSite Not Logged In Table Ble Es yew Wegcation Qata Joos Guides Window tiei 5 94 4 26 t ae9re NHE 5 803 7t jaa 367E 014 06 g 4 0 162 06 geome 015 gema 347400 96093713 Query Devices R WLVLCNPGLAEIL A08 014 2314510 3731 00 an m n 25837 20 Las 3729 10343 eR 12008 16326 40 76 42 101 31 Re na 33441 70 96093713 54298 10 96093713 30950 50 g6053713 4431340 g t9n3 2950 00 g6093713 30744 50 g amp 033713 76 26 96083713 1552 30 96033713 112 o6000013 16821 50 FEINI 68 36 96093713 1533560 96033713 21783 30 96093713 8 87 080713 1 35 nI 1673310 g amp 031713 68 35 96093713 7454 96093713 7883 96093713 437 gtom3 amp 1 03 g amp ran3 707 33 96033713 57 19 96093713 7221 96093713 26 27 GING 654 eI 121 gen 4370 9603713 5332 96093713 A 2 e fr713 R GllEQLSSGFFSPY R GLAGVENVTELKI K VMINPNSLEDVUS R VSALVK N RNLAENISR V K TCA
5. RC R3 18 Identifying Proteins and Peptides i5 ia upended eo on I Ie ee ee 20 Viewing a Saved Experimentos si eso ty ker rb Rt EY ERR Y chu cee oor 23 Understanding the ChromAlign Process Lusuuueueeseeese 21 Outlining the ChromaAlion Process 22e dae eoe dedo bx c pex bab s 28 Starting the ChromAlign Process sas ios pea VERS CREER ERR RE RP dd 29 ChromAlign First Level Alignment ite cess acte nrc s kes dere e eh 30 ChromAlign Second Level Alignment eoe d wei oy EY p wees 31 Aligning Over Different Abundance Levels llle erana 32 Aligning Over Chromalign Time Shifts scien ot sacw Eee eda oer boe ud 34 SIEVE User Guide Contents iv SIEVE User Guide Appendix B Understanding the Framing Process 0 0c eee eee eee eee eee eees 35 Aligning PAS cling Ge ieina tabbed tea a ic eO Red kis eh ono di 36 Finding the Highest Peaks cits det rre V Pope eb eR Wedd aay e 37 Appendix C Using Spotfire to View Analysis Results suuueuueeeees 39 Using the Spotfire Views oes Saws aah bach Manes ad tae dace Pa US qudd 40 Sp tfite VIEWS dd ibn quiete Dre fi LA o Aa iplc a AMNIS dot e e Qd aE A 42 Scatter Plot Views a oxacozus hace IUE whole SERRE EUER Ru rapa s ad wr 42 Protein TD View does ncn ne Ca eu veas eve xe vade iniu Ges edad et 43 Results Summary Tables acces e eem a gerat LIO PVP PEL IER 44 Thermo Scientific Preface SIEVE User Guide This guide describes how to use SIEVE to compa
6. for a SEQUEST search Values The minimum value is 0 The maximum value is 1 The default minimum value is 1 00E 98 Specifies the ID threshold for the filter criteria to improve the number of valid positive entries in the results This filter combines the peptide properties using the peptide charge and the Xcorrelation score from SEQUEST Values 1 1 5 0 1 to 10 0 2 2 5 0 1 to 10 0 3 3 7 0 1 to 10 0 4 4 5 0 1 to 10 0 Thermo Scientific 2 Setting up a Procedure Analyzing Files Tips for Improving the Process Use the following guidelines to improve the SIEVE process when comparing files To improve processing speed narrow the retention time range to the area of the chromatogram where components for example peptides and metabolites have eluted Pick the file with the best chromatogram as the first control file since all other files are compared to this reference chromatogram Analyzing Files To submit files for analysis and display initial results 1 After selecting files and setting parameters click Next on the Set Parameters window to send the raw data from the files to the ChromAlign process For a description of the alignment process see Appendix A Understanding the ChromAlign Process The Submit Analysis dialog box appears Figure 8 Options for submitting analysis E Submit Analysis Experiment name SIEVE Break points ChromAlign break point Framing break point A
7. the maximum of RT in the first control raw file Thermo Scientific SIEVE User Guide 13 2 Setting up a Procedure Setting Parameters 14 Table 1 Set Parameters dialog box values continued Parameter Retention time stop min Threshold Perform SEQUEST search Parameters file name Enter Max of frames to search pValue Xcorr vs charge state filter SIEVE User Guide LTO FT and Orbitrap default Maximum of RT in the first control raw file Definition LTO default Type the end of the retention time for the chromatogram in minutes Values The minimum value is the minimum retention time in the first control raw file The maximum value is the maximum of RT in the first control raw file Enter a value to determine the lowest MS intensity that 50 000 500000 for triggers framing LTQ FT i 1E for Values 1 to IE Orbitrap IMPORTANT If you set this value too low SIEVE might examine the total amount of frames before finding significant peaks Click Yes to search a protein database using SEQUEST Yes Yes Values Yes or No Select a SEQUEST parameter file params to be used fora SIEVE params SEQUEST search See the BioWorks User s Manual for further details The default is C Xcalibur system programs SIEVE params SIEVE params Type a maximum number of frames to be searched by 100 100 SEQUEST Values 1 to 10000 The default value is 100 Type the lowest pValue for a frame to be sent
8. to align the major features Figure 17 shows original chromatograms before the ChromAlign process begins In Figure 17 the black trace is the trace of the control sample and the lighter red trace is the test sample The ChromAlign process aligns the test sample to the control sample Figure 17 Original chromatograms Abundance 150000 200000 100000 50000 0 0 20 40 60 80 Time Min SIEVE User Guide 29 A Understanding the ChromAlign Process ChromAlign First Level Alignment 30 ChromAlign First Level Alignment SIEVE User Guide Figure 18 shows sample results from the first level ChromAlign process Some of the major peaks in the red trace are now aligned with respect to the control sample for example see the peaks at around 35 minutes SIEVE supports comparing 50 chromatograms to another 50 chromatograms totaling 100 raw data files SIEVE aligns all chromatograms to the first control file that you chose when originally selecting control and sample files Figure 18 ChromAlign first level processing Abundance 100000 150000 200000 50000 0 0 20 40 60 80 Time Min The following problems can complicate the alignment Both linear and non linear shifts in the time axis Variations in the intensity Dynamic range challenge when aligning low abundance ions in the presence of high abundance ions Note Choose a feature rich chromatographic surface a chromatogram with good signal to noise ratio t
9. 88 39 1888 Intensity Ratio 0 21895 95 CI 0 156273 0 281 pValue 6 78986e 006 ProteinID NOT YET DETERMINED DescriptionID NOT SEARCHED v lt j gt SIEVE User Guide C Using Spotfire to View Analysis Results Spotfire Views Spotfire Views Other Spotfire views include the following e Scatter Plot View Protein ID View Results Summary Table Scatter Plot View The scatter plot view is the quickest way to review the significant differences in a biomarker discovery experiment This view in SIEVE displays all of the peptide expression ratios on a single plot of ratio versus retention time Figure 27 Spotfire Scatter Plot view SIEVEI_SEARCH sfs Spotfire DecisionSite Not Logged In Scatter Plot Ble Edt sew Vieyalzation Data Tooke Guides Window Help sS 4 a2e CaOkKUMEE aoaoga lle s la a lais CT 11439 out of 1439 records visible 100 00 0 marked 42 SIEVE User Guide Thermo Scientific C Using Spotfire to View Analysis Results Spotfire Views Protein ID View Each protein can also be represented by an individual scatter plot providing a means to quickly scroll through the expression ratios for each protein identified from A to Z Figure 28 Spotfire Protein ID view SIEVEi_SEARCH sfs Spotfire DecisionSite Not Logged In Trellis Ble Edt ew visyslzation pata Took Guides Window He Pda anjou CaOkK ee Hana s E a a CT 1439 out of
10. EST search This report provides all comparative information including intensity ratios and pValues for each frame Show advanced Click this option to display a DOS window that displays the progress info procedure progress see Figure 10 3 Click Run to start the analysis and align the chromatograms If you selected Show advanced progress info in the Submit Analysis view a DOS window displays the progress of the procedure C Xcaliburlsystem programs STEVENTImeAltigner exe 16 SIEVE User Guide Thermo Scientific 2 Setting up a Procedure Analyzing Files Spotfire displays a screen similar to Figure 9 with the aligned chromatograms Figure 9 ChromAlign results SIEVEk ALIGN sfs Spotfire DecisionSite Not Logged In Scatter Plot ere wee DUM NGM a C T 39sdd a6 aeremE 5 203 3 22 Query Devices 1 18896 out of 37796 records visible 50 00 0 marked A Thermo Scientific SIEVE User Guide 17 2 Setting up a Procedure Framing the Data Framing the Data Use the following procedure to see the samples in Spotfire select relevant frames from the Spotfire Gel view and display them using the SIEVE Framing method For more information about the framing method see Appendix B Understanding the Framing Process To frame the data 1 Close the Spotfire window The Set Parameters dialog box reappears and you can adjust parameters prior to framing For information about parameter va
11. Get Started window Get Started Create a new expedient Select new experiment folder path CXXcalibu SIEVE O View evitling experiment results Select saved expenmert folder path ChromaAlign Framing Get Started page start new experiment or to view saved resuts 3 To start an experiment from the beginning select Create a new experiment To see files from a saved experiment select View existing experiment results see Viewing a Saved Experiment on page 23 for more details about this process 4 Click Browse to select the folder path In the Browse dialog box click Make New Folder 5 Name the folder and close the browser window Thermo Scientific SIEVE User Guide 7 2 Setting up a Procedure Starting a New Experiment 6 To select control and sample files for the SIEVE process click Next in the Get Started window The following window appears Figure 3 Select Data window Tip For details about a selected raw file see the lower portion of the Select Data pane Review this area to assist in selecting appropriate files for the SIEVE process 8 SIEVE User Guide Thermo Scientific 2 Setting up a Procedure Starting a New Experiment 7 Find the Xcalibur folder at the top left side of the window and click the sign to open the folders until you see the folder containing your raw files 8 Click the folder icon in this case Data to display the available raw files in the lower left wi
12. YTNHTVLPE RIGECTSDLDQLR K TORY TNHTVLPE R TNFDAEPOK V K MPAADCSEQISTA KMIPAADESEQISTS RVILENYR V ROWAMVLCNPOLAEIL VK MPAADLSEQIS TA R VAAAFPGOVOR L Mz 529 27 25 29 R WLVLCNPGLAEIL Retention Time 17 5962 20 5962 R OllEQLSSGFFSPY Intensity Ratios 121627 95 Ch 108 V APNOFNLK D pNalue 9 46500e 014 K VFADYEEYVK C Prot eielD geo13 R LKGEYPAVAATLC DeseriptioniD Gycogen Phosphor RVAAAFPGOVOR 1 K VFADYEEYMK C RWAMLCNPGLAE IL RVIFLENYR V R GlEQLSSGEFSPY R VUYPNDNEFEGK K WNDTQVWLAMP Rane LL Wo 1439 records vise 100 00 0 marked Table 4 Parameter definitions for the Results Summary table Parameter FrameID Definition A unique identifier for the frame The number indicates the order in which the frame was triggered based on the MS peak intensity For example a FrameID of 3 indicates this was the 3rd most intense MS peak that triggered the creation of a frame Average m z Average m z value Calculated from the m z start and m z stop per frame 44 SIEVE User Guide Thermo Scientific Thermo Scientific C Using Spotfire to View Analysis Results Spotfire Views Table 4 Parameter definitions for the Results Summary table continued Parameter Average time Definition Average retention time calculated from the time start and time stop per frame pValue pValue of the t test in order to provide a probability that the two distributions the log intensit
13. a cluster of peaks with the highest intensity is found triggering the creation of a frame This iterative process is similar to the way data dependent scanning operates in which the intensities of the MS peaks trigger MS MS events Using a proprietary algorithm Recursive Base Peak Framing SIEVE draws a frame around this cluster which includes peaks from both the control and treatment samples see Figure 24 The width and length of each frame depends on user defined parameters selected before starting the SIEVE process but the program suggests default values based on the type of instrument used to acquire the data Figure 24 Finding peaks intensity chromatographic time Thermo Scientific SIEVE User Guide 37 Using Spotfire to View Analysis Results The following sections describe how to use Spotfire to evaluate your experiment results and improve the process Using the Spotfire View Spotfire Views Thermo Scientific SIEVE User Guide 39 C Using Spotfire to View Analysis Results Using the Spotfire View Using the Spotfire View You can use the Spotfire view to review and fine tune the data The Spotfire view is interactive but if you make changes in the Spotfire view it does not make changes to your original experiment parameters Figure 25 shows the basic elements of the Spotfire main window Figure 25 Initial Spotfire view SIEVEi SEARCH sfs Spotfire DecisionSite Not Logged In Gel View Ble Ed
14. ame 37 files in the SIEVE process 3 introduction 2 parameter setting 57 framing process displaying DOS window 41 parameter definition 37 selecting a gel 41 G generating correlation matrix ChromAlign process 28 getting a license vii H healthy samples 2 L license getting vii SIEVE User Guide 47 Index P P parameters framing 37 setting for SEQUEST 21 parameters file name SEQUEST 20 Pearson co efficients ChromAlign process 31 peptides identifying using SEQUEST 21 viewing expression ratios 42 profiles aligning 29 Protein ID view 43 proteins identifying using SEQUEST 21 pValue selecting a relevant value 20 raw data acquiring 3 combining files 5 Results Summary Table 44 S sample files types 2 Scatter Plot View 42 selecting a gel framing process 41 selecting files 35 selecting samples for the process 2 selecting pValue parameter 20 SEQUEST defining the number of frames to search 20 identifying proteins and peptides 21 introduction 3 parameters file name 20 selecting useful gels in SpotFire 41 setting parameters 21 setting parameters for analysis 12 setting parameters for SEQUEST 21 Show advanced process info display DOS window for SEQUEST processing 16 18 21 SIEVE User Guide SIEVE comparison 2 introduction to the procedure 3 sample sets 2 SIEVE license vii SIEVE process aligning files 5 analyzing data 3 combining files 5 framing files 3 SEQUEST protein ID 3 SpotFire View De
15. arameter and is a sum from all the raw files used in the analysis Intensity_Control_ Control File number Highest intensity measurement from the specific raw file Intensity_Sample_ Sample File number Highest intensity measurement from the specific raw file SIEVE User Guide 45 Index A adjusting SpotFire query values 40 aligning creating an optimal path 31 files in the ChromAlign process 28 files in the SIEVE process 3 profiles 29 analysis setting parameters 12 analyzing data 3 base peak information aligning profiles 29 C changing SpotFire query values 40 ChromAlign process base peak information 29 defining an optimal path chromatographic surfaces 28 first level alignment 28 generating correlation matrix 28 introduction 2 optimal path 31 Pearson co efficients 31 starting 28 combining files in the SIEVE process 3 raw data files 3 Contacting x control files selecting 35 control samples 2 data analyzing 3 selecting relevant subsets in Spotfire 41 sorting in Spotfire 41 Thermo Scientific data files minimum number supported 2 total number supported for comparison 2 defining parameters for Framing 37 defining number of frames to search in SEQUEST 20 disease samples 2 DOS window display for SEQUEST processing 16 18 21 F FFT aligning profiles 29 files selecting 35 filtering data using SpotFire Range Sliders 41 framing chromatographic shifts 36 comparing files 36 creating a fr
16. aw file IMPORTANT If you set this value too low SIEVE might examine the total amount of frames before finding significant peaks m z stop Type the maximum m z value to be analyzed for each file Maximum m z of full range in the first control Values The minimum value is the minimum m z of full range aw file in the first control raw file The maximum value is the maximum m z of full range in the first control raw file Frame m z width Type a value to define the width of each frame 1 5 0 02 SIEVE displays a suggested value Values 0 01 to 50 0 Frame time width Type a value to define the width of each frame in minutes 2 5 2 5 min Decrease this number for narrower peaks increase this number for wider peaks SIEVE displays a suggested value Values 0 1 to 10 0 The default value is 2 5 IMPORTANT Ifyou set this value too low SIEVE might examine the total amount of frames before finding significant peaks Search peak width This value represents a percentage of the width of each frame 30 30 96 starting from the center to define the area where SEQUEST data files dtas are generated for a search SIEVE display a suggested value Values 10 to 100 The default value is 30 Retention time start Type the start of the retention time for the chromatogram in Minimum of RT in the min minutes first control raw file Values The minimum value is the minimum retention time in the first control raw file The maximum value is
17. chromatograms to be aligned with respect to the first control chromatogram Step 1 Step 1 of the ChromAlign process produces a crude alignment of the two Align chromatograms roughly using Fast chromatographic surfaces using a fast fourier transform algorithm to Fourier Transform FFT algorithm maximize the overlap of the chromatographic surfaces FFT A first control sample FFT B all other chromatograms FFT FFT B xFFT A Crudely aligned B Step 2 In step 2 SIEVE uses the full MS scan information to fully align all Generate correlation matrix C A B components major as well as minor components of the samples of the chromatographic surfaces SIEVE uses dynamic programming to maximize the sum of all correlation co efficients of peaks between time 0 and the end of the chromatogram Step 3 SIEVE completely aligns the chromatographic surfaces Create dynamic programming optimal path in C A B and provide a 28 SIEVE User Guide Thermo Fisher A Understanding the ChromAlign Process Starting the ChromAlign Process Starting the ChromAlign Process Thermo Scientific After you submit the data for analysis SIEVE aligns the chromatographic profiles using the base peak information only using fast fourier transforms FFTs to provide a transformation based correlation analysis between the two chromatograms see Step 1 This alignment produces a correlation array showing exactly how much to shift the various chromatograms
18. data subsets of interest Use the left and right arrows to change the lower and upper limit of the range displaying and selecting only records with values within the chosen range Labels above the slider indicate the selected span The slider automatically jumps to values in the data set not necessarily the visible or selected records Click and drag the yellow portion of the range slider to pan the selected range Observing the response of the other sliders to such a sweep can give some interesting clues about the correlation between parameters in the data set For more information see Spotfire Help The View Details on Demand text option from the Spotfire View menu default displays data in the lower right side of the Spotfire display After selecting a frame from the gel view use this data to decide whether the frame is useful for further examination Click a gel in the Spotfire window to select it a circle surrounds it and display the following Reconstructed ion chromatograms for that frame The mass to charge ratio around which the frame was centered the ratio of peak intensities between the control and treatment samples The confidence interval The pValue indicating whether the ratio was statistically significant Figure 26 Spotfire detail for a frame specified by pValue Details on Demand Html RIC Jl Y ID Candidates Accepted ID Hits Rejecte _ M Z 1080 22 1080 24 Retention Time 36 68
19. detail information display RIC Summary ID Candidates Accepted ID Hits Rejected ID Hits MS MS SEQUEST Hit ID Criteria About Reconstructed Ion Chromatogram Intentsity N eo o x 2 9K ae Lj pat ne a 553 37 603 37 853 38 103 38 353 38 603 38 853 39 103 39 353 39 603 39 853 Time minutes Control A1 ControlA2 Control A3 9 Control A4 Control_AS Sample_61 54mple B3 95Sample B4 Sample B5 FRAME 1083 TIME 37 3535 39 68535 MASS 1169 08 1169 1 RATIO 6078 5 95 CI 6589 85 18746 6 pVALUE 0 000180416 sample B2 Frame List Note SIEVE does not display raw file names when you select more than 18 control and sample raw files 9 control and 9 sample files 4 To exit the SIEVE application click Close SIEVE in the SIEVE window Thermo Scientific 2 Setting up a Procedure Viewing a Saved Experiment Viewing a Saved Experiment As you follow the process to create and view a new experiment SIEVE automatically saves the files that result from the different stages of the process To view a saved experiment 1 To see these results click Get Started and click View existing experiment results Figure 14 Get Started window Get Started Create a new experiment Select new experiment folder path Select saved expenmert folder path C Xcalibu SIEVE ChromAlign Framing Ge Started page start a new experiment or to view saved results Thermo Scie
20. dvanced progress V Show advanced progress info Thermo Scientific SIEVE User Guide 15 2 Setting up a Procedure Analyzing Files 2 Specify a new experiment name and define break points for various steps so that you can evaluate the intermediate results and make changes See the following table for information about the options SIEVE displays interactive windows for any break points you select If you don t select any break points SIEVE finishes the entire process without interruption Table 2 Submit Analysis parameters Experiment Type the name of the experiment using the following criteria Name Do not type the name of an existing experiment Do not us a name with spaces or special characters Usean underscore to separate parts of the file name If you enter an existing experiment name or an invalid file name SIEVE displays an error message ChromAlign Select this option to pause after the ChromAlign process so that you break point can evaluate the results This option opens a Spotfire display after the chromatograms in all control and sample files have been aligned Use the display to view alignment and to determine whether to continue or to start over with different files Framing break Select this option to pause the procedure after the framing process so point that you can evaluate the results This option opens a Spotfire report that includes all framing information before you start a SEQU
21. eset unless instructed to do so by an authorized Thermo Fisher Scientific representative To geta license code 1 Install the SIEVE software by clicking the SIEVE install icon 2 Follow the prompts to install the software 3 To start SIEVE choose Start gt All Programs gt Xcalibur gt STEVE or click the SIEVE icon on your computer desktop If you do not have a current license the following screen appears SIEVE License Control The SIEVE software has not been licensed There are two types of licenses available 30 day Evaluation Version Full Single Version To enable SEQUEST search you must obtain a valid license for Bioworks If you have a license please enter it now The license change wil take effect the nest time you run the SIEVE software To obtain a license please email your customer information product information the type of license desired and the license to licenses ms GXthermo com Thermo Scientific SIEVE User Guide vii Preface Getting a License 4 Click Change License to display the license dialog box SIEVE License The SIEVE software has not been licensed There are two types of licenses available 30 day Evaluation Version Full Single Version To enable SEQUEST search you must obtain a valid license for Bioworks If you have a license please enter it now The license change will take effect the next time you run the SIEVE software To obtain a license please emai your custo
22. fferent Abundance Levels ChromAlign produces perfect alignment of the 100 fmol trace with the 1000 fmol trace Figure 21 Chromatograms after alignment e ce ce ce re LI ce ce ce ce e a o c o c lt e ce ce e re ce 49 50 51 52 53 Time Min Thermo Scientific SIEVE User Guide 33 A Understanding the ChromAlign Process Aligning Over Chromalign Time Shifts Aligning Over Chromalign Time Shifts See Figure 22 to understand the complexity of the alignment process by plotting the chromatographic time warping function Although this plot uses data from a different set of control and sample files it demonstrates the complexity of the alignment effort caused by chromatographic shifts The average alignment score for the alignment in this figure is 0 53 Figure 22 Chromatographic time warping required to create alignment Time Warp 100 200 300 400 500 Time Min 34 SIEVE User Guide Thermo Fisher Understanding the Framing Process Thermo Scientific To understand the framing process in SIEVE consider that biomarker or differential expression experiments require comparing at least two sets of raw data files each representing two distinct biological states A compared to B however to be statistically meaningful a well designed biomarkers study should aim to compare multiple bioreplicates from control and treatment disease sets each analyzed ideally multiple times as technical replica
23. formation about using the Spotfire view see Appendix C Using Spotfire to View Analysis Results For further information about seeing and manipulating your data with Spotfire see Spotfire Help Thermo Scientific SIEVE User Guide 19 2 Setting up a Procedure Identifying Proteins and Peptides Identifying Proteins and Peptides After SIEVE completes the framing process use the following procedure to send the data to SEQUEST to identify proteins and peptides After using SEQUEST to perform a database search on the frame Spotfire displays a table for that frame with information about the protein ID and description peptide sequence XCorr charge state and peptide identification To send the data to SEQUEST 1 Close the Spotfire report The following window appears Figure 11 SEQUEST parameters Criteria for SEQUEST Search Select SEQUEST Parameter fie 9 BIRARTSINET Max Softener 100 pValue threshold 1 00608 Complete parameter values as follows Table 3 SEQUEST parameters Select SEQUEST To select a SEQUEST parameter file params to be used for the parameter file search click Browse The default is SIEVE params Max of frames Enter the maximum number of frames to search Values 1 10000 The default value is 100 pValue threshold Type the lowest pValue for a frame to be sent for a SEQUEST search Values 0 0 to 1 0 The default value is 1 00E 95 20 SIEVE User Guide Thermo Scientific 2
24. ic 441340 ING 2883 00 96093713 30744 50 ge083713 75 26 g6083713 15529 30 96093713 112 g 0580 13 1685 50 gta 6836 6033713 15335 60 96083713 21763 30 96083713 887 96089713 HSIN 1673310 96083713 68 35 ge033713 7454 96033713 78 83 96083713 437 MON 0 get 3707 33 g6033713 57 18 6093713 7221 g6083713 BI AGING 544 on 121 96m3n3 4370 g amp 093713 5332 96083713 AG F2 ORIN RWLVLCNPGLAEIL R GliEQLSSGFFSPY R GLAGVENVTELKI K VHINPNSLEDVOS RVSALYK N RNLAENSR V K TCAYTNHTVLPE RiGECTSDLDQUR K TCAYTNHTVLPE R TNFDAEPOK V K MPAADLSEQISTA K MPAADLSEQISTA RVIFLENYR V RWAMLCNPGLAEIL K MPAADLSEQIS TA R VAMFPGONVOR L R WLMLCNPGLAEIL ROGMEQLSSGFFSPY KAPNOFNUK D K VFADVEEYW C RLKOEYFWAATLC R VAMAFPGOVOR L K VFADYEEYWC C RWLALCNPGLAE IL RVrLuENTR V R GIEQLSSGFFSPY R VLYPNDNEFEGK KWVDTQWLAMPs AVANT Details on Demand imi alx Mz 599 27 29 29 Retention Tise 17 8962 20 9962 Intensity Ratios 121627 95 CI 206 pValue 9 465080 014 Proteini eons DescriptionlD QycogemPhospherylae LLL WH 1439 records viste 100 00 0 marked SIEVE User Guide 21 2 Setting up a Procedure Identifying Proteins and Peptides 22 SIEVE User Guide This table is completely interactive For specific information about a frame click the entry Click on a frame in any of the views for example a frame in the Gel Spot view or any cell in the Table view to display this view Figure 13 SIEVE
25. ies of the control and treatment are different A smaller pValue indicates a more statistically significant difference was measured Ratio The average of the log peak intensities for each replicate in each group LSample Average IPeak1 IPeak2 Sample and LControl Average IPeak1 IPeak2 Control The ratio is the ratio of these numbers LSample LControl UpperBound The 95 confidence interval for this ratio based upon the statistics derived from the replicates The 95 confidence interval is two numbers a lower limit and an upper limit within which there is a 95 probability that the true ratio resides LowerBound Protein Description The 95 confidence interval for this ratio based upon the statistics derived from the replicates The 95 confidence interval is two numbers a lower limit and an upper limit within which there is a 95 probability that the true ratio resides An ID number for identified proteins Description of the protein or the state of the frame if no protein is identified Peptide Name of the identified peptide XCorr The SEQUEST cross correlation score of the top candidate peptide or protein Cross correlation the multiplication of two signals averaged over a time interval Charge The charge of the peptide identified from the database search MS2 The number of ms2 scans that belong to the frame This number is dependent on the search width SIEVE p
26. ignificantly decreases the time spent identifying proteins and increases the throughput of complex sample sets Step 6 Review Interactive Results SIEVE provides interactive results using several different Spotfire plots specifically chosen to maximize the information extracted from these types of biomarker discovery experiments Breakpoints after ChromAlign Recursive Base Peak Framing and SEQUEST provide the option of checking intermediate results before proceeding or adjusting parameters 4 SIEVE User Guide Thermo Scientific Setting up a Procedure Thermo Scientific The following sections describe setting up and running a procedure for SIEVE using two sets of raw files Starting a New Experiment Setting Parameters Analyzing Files Framing the Data Identifying Proteins and Peptides Viewing a Saved Experiment SIEVE User Guide 5 2 Setting up a Procedure Starting a New Experiment Starting a New Experiment To start a new experiment and select data for the analysis 1 To start SIEVE choose Start gt All Programs gt Xcalibur gt STEVE or click the SIEVE icon on your computer desktop The following window appears Figure 1 SIEVE home page SIEVE 1 1 0 Automated Label Free Differential Expression Software 6 SIEVE User Guide Thermo Scientific 2 Setting up a Procedure Starting a New Experiment 2 Click Get Started to display experiment options The Get Started window appears Figure 2
27. l data up to 100 data files is analyzed in the common multivariate space described in Step 2 Consequently SIEVE must correct for any chromatographic variation across the samples under analysis For this purpose SIEVE employs a unique chromatographic alignment algorithm called ChromAlign Step 4 Frame the data SIEVE combines each raw file on a 3D plane of m z versus retention time versus intensity SIEVE automates the process of determining differential expression by employing a proprietary algorithm called Recursive Base Peak Framing This algorithm statistically compares the intensity of the peaks within each frame areas in the m z by chromatographic time plane where differential expression is likely to have occurred The primary plot generated by SIEVE within Spotfire displays retention time versus 7 z Each blue rectangle represents a single frame The consolidated plot is made up of several hundred to several thousand frames the darker of which indicate a more statistically significant difference Thermo Scientific SIEVE User Guide 3 1 Overview to SIEVE About the SIEVE Procedure Step 5 Analyze the data using SEQUESTto Select SEQUEST to perform a database search only on frames identify proteins determined to have confident pValues This step identifies peptides and proteins that might represent differential expression Using SIEVE to pre filter data greatly reduces the number of spectra that need to be searched s
28. lues see Setting Parameters on page 12 2 Click Next to frame the data If you selected Show advanced progress info in the Submit Analysis view a DOS window displays the progress of the procedure IMPORTANT Stay in the SIEVE window when the system is processing If you click another window or change programs you might disrupt the processing Spotfire opens displaying the primary plot Primary plot Generated by SIEVE within Spotfire this plot represents frames colored by the pValue to indicate significant differential expression within the frame Consolidated plot Made up of several hundred to several thousand frames this plot shows frames of various colors where darker frames gels indicate a more statistically significant difference 18 SIEVE User Guide Thermo Scientific 2 Setting up a Procedure Framing the Data 3 To filter down to frames of interest move the pValue slider in the Query Devices section on the right side of the window If you are looking for a specific ratio filter by pValue retention time range m z range or even intensity ratios Low pValues indicate significant changes Figure 10 Spotfire view with filtered pValues SIEVE _FRAME sfs Spotfire DecisionSite Not Le ogged In bel View Ble Edt yew Veystetion puta Tools Guides Window Heb e x sou dam ATCA ajana jeej jaaj Query Devices 5 14403 out of 4403 records visible 100 00 0 marked For more in
29. mer information product information the type of license desired and the license to licenses ms thermo com Note Do not click Reset unless instructed to do so by an authorized Thermo Fisher Scientific representative 5 Highlight the license key in the License text box 6 Press CTRL C to copy the key to the Windows clipboard 7 Send an e mail message to licenses ms thermo com a Type license request in the subject line of the e mail and paste the license key into the body of the e mail b Provide your customer information c Locate the bar code on the back of the SIEVE CD jewel case Type the serial number that appears below the bar code into the body of the e mail Thermo Fisher Scientific Customer Support sends you a new license code To install the new license code go to the next section viii SIEVE User Guide Thermo Scientific Preface Safety and Special Notices Installing a New License Code Once you have received your new license code install it as follows To install the license code 1 To start SIEVE choose Start gt All Programs gt Xcalibur gt STEVE or click the SIEVE icon on your computer desktop 2 Click Change License to display the SIEVE License dialog box The SIEVE License dialog box appears 3 In the License text box type the new license number and click Set 4 To accept the change in license click Yes 5 To close the SIEVE Browser License dialog click Close Note Do not ch
30. ndow Figure 4 SIEVE folder display Thermo Scientific SIEVE User Guide 9 2 Setting up a Procedure Starting a New Experiment 9 To select control files raw extension only from the file list in the lower left side of the screen click on the file names Use the CONTROL key to select more than one file at a time Figure 5 Selecting raw files from the SIEVE folder display Creator ID LTO Fiename C Xcalburexsmoles i SIEVE data Contol_Al RAW Version 57 Creation Date 1 11 2006 10 46 04 AM it New File Falte In Acquisition False View the window at the bottom of the screen to find additional raw file information Tip To improve reliability select control and sample files that have been acquired on the same instrument with the same length of gradient Do not select the same file for both control and sample Tip To achieve optimal alignments choose a feature rich chromatographic surface as the first control sample a chromatogram with good signal to noise ratio 10 SIEVE User Guide Thermo Scientific 2 Setting up a Procedure Starting a New Experiment 10 Click Add to add the files to the Control folder 11 To add sample files click the Sample tab Select raw files from the file list and click Add to add the files to the Sample folder Figure 6 Adding sample files Control Sample Sample B1 RAW Sample B2 RAW Sample B3 RAW Sample B4 RAW Sample B5 RAW Thermo Scientific SIEVE Use
31. nment for data review Use SIEVE to identify protein biomarkers that indicate the presence of disease or identify potential targets in the drug discovery and development process Thermo Scientific 1 Overview to SIEVE About the SIEVE Procedure About the SIEVE Procedure The following section describes the SIEVE procedure For specific information on each step see Chapter 2 Setting up a Procedure Step 1 Acquire raw data Discovery experiments for global biomarker often consist of thousands of complex MS and MS data SIEVE accommodates both high resolution and low resolution data including data from the LTQ XL linear ion trap mass spectrometer as well as the hybrid LTQ Orbitrap and LTQ FT Ultra instruments Step 2 Combine multiple data An effective biomarker experiment requires the comparison of at least two raw data files but to be statistically meaningful the experiment must include several control samples compared to several treatment disease samples in the form of technical replicates and bio replicates SIEVE supports comparing 50 chromatograms to another 50 chromatograms totalling 100 raw data files To facilitate this comparison SIEVE presents the data in one consolidated view that you can quickly view for differences SIEVE can combine and compare from 2 to 100 data files Step 3 Align Chromatograms Chromatographic alignment is an important step in the SIEVE pipeline since all the experimenta
32. ntific SIEVE User Guide 23 2 Setting up a Procedure Viewing a Saved Experiment 2 To locate the top level folder with experiment results in this case C Xcalibur SIEVE click Browse Select that folder in the browse window and click OK to display the list of possible results A red x indicates a SIEVE process that was not completed Figure 15 View Experiment Results window View Experiment Results Select saved experiment folder path C XKealiburi SIEVE Get Stated Epai pa tesults dur rey opm resulls ft ra Mp Close SIEVE m A A ChromAlign Click on an icon to view specified results or Get Started to start an experiment 24 SIEVE User Guide Thermo Scientific 2 Setting up a Procedure Viewing a Saved Experiment 3 To see parameters and other details for the experiment click an icon or the experiment name to see the results Figure 16 Experiment Parameters window experiment lypes AvsB mzStart 400 00 2000 00 FrameMzwidth 0 02 RetentionTimeStart 0 00 RietentonTimeStops B4 SG FrameTimeWidth 2 50 threshokd SO0000 maxpvate 0 00000001 maxFramesT ol 2000 SearchPeakWidth 0 30 h strearmer fiberstimgs Full ms Ditectones intermediates Intermediate resullie Results sequeste Sequest IDthveshiold xcon C1 1 5 4 To return to the View Experiment Results window click Back To exit the SIEVE application click Close SIEVE Thermo Scientific SIEVE User Guide 25
33. o achieve optimal alignments Figure 18 shows the alignment of two chromatographic runs of a digested nine protein mixture horse myoglobin bovine serum albumin chicken egg lysozyme chicken egg ovalbumin bovine carbonic anhydrase bovine f casein horse cytochrome C bovine a lactalbumin and rabbit glyceraldehyde 3 phosphate dehydrogenase Thermo Fisher A Understanding the ChromAlign Process ChromAlign Second Level Alignment ChromAlign Second Level Alignment The second step of the ChromAlign process refines the alignment of the roughly aligned chromatograms ChromAlign generates a correlation matrix between full MS scans of the two chromatograms populated by the Pearson co efficients obtained from the full MS data Then it assigns artificially large values to the correlations between the first scans and the last scans respectively see Step 2 ChromAlign then aligns the first and last scans connecting these points to maximize the sum of the correlation co efficients see Step 3 Dynamic programming generates the optimal path maximum sum of the correlation co efficients to yield a chromatographic time warping function see Aligning Over Chromalign Time Shifts Applying this function to the chromatographic surface of the sample produces full alignment of both data sets see Aligning Over Different Abundance Levels on page 32 After final alignment SIEVE displays the optimal path in the form of the aligned chromatographic
34. oose Reset at this point or the license software invalidates the license P you just received Safety and Special Notices Make sure you follow the precautionary statements presented in this guide The safety and other special notices appear in boxes Safety and special notices include the following CAUTION Highlights hazards to humans property or the environment Each CAUTION notice is accompanied by an appropriate CAUTION symbol IMPORTANT Highlights information necessary to prevent damage to software loss of data or invalid test results or may contain information that is critical for optimal performance of the system Note Highlights information of general interest Tip Helpful information that can make a task easier Thermo Scientific SIEVE User Guide ix Preface Safety and Special Notices Contacting Us There are several ways to contact Thermo Fisher Scientific To contact Technical Support Phone 800 685 9535 Fax 561 688 8736 E mail TechSupport C MS thermofisher com Knowledge base www thermokb com Find software updates and utilities to download at www mssupport thermo com To order products or to get product updates Phone 800 532 4752 Fax 561 688 8731 Web site www thermo com finnigan To suggest changes to documentation or to Help Fill out a reader survey online at www thermo com Icms techpubs Send an e mail message to the Technical Publications Editor at techpubs finnigan lcms thermofi
35. r Guide 11 2 Setting up a Procedure Setting Parameters Setting Parameters To set parameters 1 After selecting files for the analysis click Next to display the Set Parameters dialog box SIEVE populates each parameter in an edit box with recommended default values based on the instrument used to acquire the first raw file listed in the Control group You can modify these values from the original default 2 Change parameters as needed Figure 7 Set Parameters dialog box Set Parameters Intensity Chromatographic time tion time start min SEQUEST Perform SEQUEST search YES ONO Parameters fle name CNXXcalibu paramsNSIEVE params Enter Max of frames to be searched 100 pValue 1 00e 08 Xcor vs chage state filter 41 15 2 25 3 37 44 SIEVE uses these parameters to define a frame to determine the MS intensity threshold and to define the limits of the experiment 12 SIEVE User Guide Thermo Scientific 2 Setting up a Procedure Setting Parameters Table 1 Set Parameters dialog box values LTO FT and Parameter Definition LTO default Orbitrap default m z start Type the minimum m z value to be analyzed for each file Minimum m z of full range in the first control Values 400 The minimum value is the minimum m z of full saw file MS range in the first control raw file The maximum value is the maximum m z of full MS range in the first control r
36. re multiple LC MS data sets to determine the statistically significant differences between samples and identify the corresponding peptides and proteins To help you make the most of SIEVE review the following topics Viewing the Help System Requirements Getting a License Safety and Special NoticesContacting Us Contacting Us Viewing the Help In addition to this guide Thermo Scientific provides Help that you can access from the software Thermo Scientific SIEVE User Guide v Preface System Requirements vi System Requirements SIEVE User Guide The minimum hardware requirements for SIEVE 1 1 0 are as follows 2 GHz processor 1 GB RAM 40 GB hard disk formatted with NTFS CD ROM drive Display resolution 1280 x 1024 XGA with small standard fonts To install and use SIEVE 1 1 0 requires the following software Windows XP with SP2 Microsoft Office XP Professional SP2 BioWorks 3 3 1 Xcalibur 2 0 5 or higher Microsoft Internet Explorer 6 0 Thermo Scientific Preface Getting a License Getting a License To get a license for SIEVE 1 Install the SIEVE software according to the instructions on the CD cover 2 Get a new license code from Thermo Fisher Scientific 3 Install the new license code Getting a New License Code The following instructions describe using e mail to get a license code If you do not have access to e mail you can get a license by fax Note Do not click R
37. sher com x SIEVE User Guide Thermo Scientific Overview to SIEVE SIEVE software performs semi quantitative differential analyses of sample populations by comparing spectral information from LC MS analyses of control healthy and disease treatment samples Any changes between the two sample sets indicate a differential protein expression Elements from raw files that show significant statistical differences are sent for a SEQUEST database search to determine peptide and protein identification The following sections provide a high level overview of SIEVE Understanding SIEVE About the SIEVE Procedure Thermo Scientific SIEVE User Guide 1 1 Overview to SIEVE Understanding SIEVE Understanding SIEVE 2 SIEVE User Guide SIEVE is an automated software package for the label free semi quantitative differential expression of proteins and peptides It performs comparative analyses of sample populations by comparing the raw spectral information from LC MS analyses of control healthy and disease treatment samples to determine if there are changes among the two sample sets which may indicate differential protein expression SIEVE provides a statistically rigorous tool for analyzing the data acquired in protein biomarker discovery experiments SIEVE can analyze as many as 100 LC MS data files in an experiment that compares 50 sample files to 50 control files in a single operation It can also compare a minimum of four files select t
38. t yew Visysizshon Data Tools Guides Window Help 9d BSH roae9rr UHN B ADS SE aa Query Devices FramelD 1 Query Range Sliders BetaCasen_BOVINE F Catalase BOVINE fv DOES NOT PASS ID CRI Iv Glycogen Phosphorylase fV NO MS MS SPECTRA P fv NOT SEARCHED IDetails on Demand a x RIC Summer 1D Candidat M 2 1269 0 Retention Time 25 014 50 Intensity Ratio 2 11315 SS ij Aveszge Tere 7 Get View Scatter Piot Tabie Teele lt 46 58 1663 07 LLL 1439 out of 1439 records visible 100 00 0 marked Gel view Table view Frame details Scatter plot view Protein ID view You can keep several Spotfire windows open at the same time up to three but if you click Get Started SIEVE closes all the Spotfire windows You can do any of the following Adjust query values to exclude frames that do not match search criteria For example to adjust the slide range for pValue to include pValues that are closer to 0 slide the rightmost arrow away from 1 0 To display frame information on the Spotfire page select a frame by clicking on a gel in the Spotfire Gel view View the data in several views see Spotfire Views on page 42 40 SIEVE User Guide Thermo Scientific C Using Spotfire to View Analysis Results Using the Spotfire View Spotfire Range Sliders Frame Details Gel Cell Thermo Scientific Use the Range sliders to select a range of values to filter the data or to select
39. tails on Demand option 41 views introduction 39 Protein ID View 43 44 Scatter Plot View 42 SpotFire Range Sliders 41 SpotFire view adjusting query values 40 fine tuning data 40 how to use 40 SpotFire Views 42 starting ChromAlign process 28 V View Details on Demand displaying DOS window for Framing 41 selecting useful gels for SEQUEST 41 SpotFire 41 viewing m z value 44 protein identification 44 proteins 45 pValue 44 rate of change 44 XCorr 44 Thermo Scientific
40. tes Furthermore presenting the results of the typically large volume of generated data in one consolidated view improves the differential analysis This discussion of the framing process covers the following topics e Aligning Peaks Finding the Highest Peaks SIEVE User Guide 35 B Understanding the Framing Process Aligning Peaks Aligning Peaks In this simplified representation of an experiment comparing three raw files Controls to three raw files Treatments SIEVE first consolidates the spectra in a multivariate space Retention time versus m z versus full scan ms intensity see Figure 23 Each of the control samples is represented by green spectral peaks on the map each of the treatment samples is represented by red spectral peaks on the map In a real set of samples thousands of peaks would populate this plane Figure 23 SIEVE consolidates the spectra intensity Vis chromatographic time Notice that there are green outlying peaks to the left of each of the three main clusters These peaks represent chromatographic shifts in retention time requiring that the peaks first be aligned using the ChromAlign process 36 SIEVE User Guide Thermo Fisher B Understanding the Framing Process Finding the Highest Peaks Finding the Highest Peaks After alignment is complete framing begins with the most intense MS peak To find the first most intense peak imagine a flat plane being lowered vertically over the 3D plot until
41. times and provides the alignment score the average of the correlation matrix along the generated optimal path Figure 19 shows the aligned results for the same chromatograms where all components of the nine protein mixture are fully aligned Figure 19 ChromAlign second level processing c c c c c N c c c ce LO oO e c c e c He c c c D gt Q lt c c c ce LO c 0 20 40 60 80 Time Min This example demonstrates good alignment after the ChromAlign process even though the alignment is not a simple static shift Thermo Scientific SIEVE User Guide 31 A Understanding the ChromAlign Process Aligning Over Different Abundance Levels Aligning Over Different Abundance Levels Biomarker discovery detects low abundance components in the presence of high abundance components that remain essentially unchanged between the controls and samples The following example shows the alignment of low abundance components in the presence of high abundance components Figure 20 shows reconstructed ion chromatograms of a horse myoglobin peptide added to the nine protein mix at three different concentrations 500 fmol red 250 fmol green and 100 fmol blue and compared to the control sample 1000 fmol black Figure 20 Original reconstructed ion chromatograms 100000 150000 Abundance 50000 49 50 51 52 53 Time Min 32 SIEVE User Guide Thermo Fisher A Understanding the ChromAlign Process Aligning Over Di
42. tract between Thermo Fisher Scientific Inc and a purchaser This document shall in no way govern or modify any Terms and Conditions of Sale which Terms and Conditions of Sale shall govern all conflicting information between the two documents Release history For Research Use Only Not regulated for medical or veterinary diagnostic use by U S FDA or other competent authorities Contents Thermo Scientific Chapter 1 Chapter 2 Appendix A Prelate PUE v SIEVE User Guides s bora ipo S ERE ood eee EE EEEE v Viewing the Helps ax dat uere dotes de ee dona meson anne p ap deos oa ess x v System Requirements 00 cee eee ete en vi Getting a License ie dreie sida cea LRG Mae bates ow band a eaae bes vii Getting a New License Cade sd ed doe ye Von hc Pte ele ye cee PR vii Installing a New License Code ii iios ecbketk more tk e rore ee oe bac e ix Safety and Special Notices cress aio a pp ORE RARE ew nares e ro iR ix Contacting Us ese galas e eese dera e ue dude xu relie Gee eens x Overview to SIEVE ccoin nanon manyan aaa dui als a bob a a 1 Understanding SIEVE eia aceite racio aon tne a E 2 About the SIEVE Draeedute ce eed eco kw EY ER acto OUR cR CK E 3 Setting up a Procedure Luuuuuuuesuesseeeeseeeeeeeeeeeeee 5 Starting a New Experiment nen 6 Setting Parameters cess esee eher wd s CE HU CEP a e e 12 Analyzing Files pedcs T c rEETETTTEN 15 Framing the Data 5 a tito oo aee doo Re E Oo E o EOS
43. wo control files and two sample files so that the pValue is valid SIEVE employs a Chromalign algorithm that allows comparison of similar chromatographic surfaces From here it uses a proprietary iterative process called Recursive Base Peak Framing to find statistically meaningful differences For more information about the ChromAlign procedure see Appendix A Understanding the ChromAlign Process For more information about the framing process see Appendix B Understanding the Framing Process To identify proteins SIEVE performs a comparative analysis prior to a SEQUEST database search To identify peptides and proteins send elements from the raw data files that show significant statistical differences for a SEQUEST database search SIEVE maximizes computational time and resources by providing the option to search only those features that have shown statistically significant change accelerating the process of finding potential biomarkers SIEVE uses the MS intensities from the raw LC MS data without the need to manipulate or model peaks to find statistical differences The process is label free using no isotopic tags or labels of any kind SIEVE calculates a pValue to indicate the statistical significance of the expression ratio of each putative biomarker providing more confidence inthe results SIEVE displays intermediate and final results using the Spotfire DecisionSite software a powerful interactive visual enviro
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