GPS Explorer Software For Protein Identification Using the Applied Biosystems 4700 Proteomics Analyzer

Transcription

1 GPS Explorer Software For Protein Identification Using the Applied Biosystems 4700 Proteomics Analyzer Getting Started Guide

2 GPS Explorer Software For Protein Identification Using the Applied Biosystems 4700 Proteomics Analyzer Getting Started Guide July 18, :22 pm Wallace_title.fm

3 Copyright 2002, Applied Biosystems. All rights reserved. For Research Use Only. Not for use in diagnostic procedures. Information in this document is subject to change without notice. Applied Biosystems assumes no responsibility for any errors that may appear in this document. This document is believed to be complete and accurate at the time of publication. In no event shall Applied Biosystems be liable for incidental, special, multiple, or consequential damages in connection with or arising from the use of this document. Applied Biosystems is a registered trademark of Applera Corporation or its subsidiaries in the U.S. and certain other countries. AB (Design), Applera, and Explorer are trademarks of Applera Corporation or its subsidiaries in the U.S. and certain other countries. ICAT is a trademark of the University of Washington in the U.S. and certain other countries, exclusively licensed to the Applied Biosystems Group of Applera Corporation. Dell is a trademark of Dell Computer Corporation. Windows NT, Microsoft Access, and Visual Basic are registered trademarks of Microsoft Corporation. Microsoft and Windows are trademarks of Microsoft Corporation. Mascot is a registered trademark of Matrix Science Ltd. Pentium is a registered trademark of Intel Corporation. All other trademarks are the sole property of their respective owners. Printed in the USA, 06/2002 Part Number Rev. B July 18, :22 pm Wallace_title.fm

4 Contents How to Use This Guide Chapter 1 Introduction to the GPS Explorer Software Workflow for Identifying Proteins Logging In and Out of GPS Explorer Software Chapter 2 MS Only for Protein ID (PMF) Create a Sample Set Create an Analysis Prepare and Submit Samples for Processing Monitor Job Processing View Results Chapter 3 MS/MS Only for Protein ID Create a Sample Set Create an Analysis Prepare and Submit Samples for Processing Monitor Job Processing View Results Chapter 4 Combining MS and MS/MS Spectra for Protein ID Create a Sample Set Create an Analysis Prepare and Submit Samples for Processing GPS Explorer Getting Started Guide July 18, :22 pm, Wallace_GSTOC.fm iii

5 Monitor Job Processing View Results Chapter 5 Protein ID by Sample Set (MS/MS Spectra Only) Create a Sample Set Create an Analysis Prepare and Submit Samples for Processing Monitor Job Processing View Results Chapter 6 Creating Reports Design a Report Report a Sample Associated by Well Including All Wells Including Well Range Report a Sample Associated by Set Export Reports Appendix A Displaying Information Set the Well Scale A-2 Display User-Defined Sample Information A-2 Display User-Defined Columns A-2 Import Column Information A-3 Customize Well-Color Display A-5 Appendix B Managing the GPS Explorer System Configure the Desktop B-2 Configure the Instrument B-4 Manage Database Functions B-5 Manage Users B-6 iv July 18, :22 pm, Wallace_GSTOC.fm GPS Explorer Getting Started Guide

6 Appendix C Warranty and Service Information Limited Product Warranty C-2 Damages, Claims, Returns C-5 GPS Explorer Getting Started Guide July 18, :22 pm, Wallace_GSTOC.fm v

7 vi July 18, :22 pm, Wallace_GSTOC.fm GPS Explorer Getting Started Guide

8 How to Use This Guide Purpose Audience Conventions The GPS Explorer Getting Started Guide is an introduction to the GPS Explorer software. This guide describes how to use the software by presenting four different examples of experiments supported by GPS Explorer software. (For detailed information on using the software, see the GPS Explorer software online Help.) This guide is intended for novice users of the GPS Explorer software. This guide uses the following conventions: Bold indicates user action. For example: Type 0 and press Enter for the remaining fields. Italic text denotes new or important words and is also used for emphasis. For example: Before analyzing, always prepare fresh matrix. A right arrow bracket (>) separates successive commands in a command sequence, beginning with a drop-down or shortcut menu. For example: Select File > Open Database GPS Explorer Getting Started Guide July 18, :22 pm, Wallace_GS_howto.fm vii

9 How to Use This Guide Services and Support Send your comments For Technical Support and ordering information, dial (North America only) or , or fax questions to To access the Applied Biosystems web site, go to: At the Applied Biosystems web site, you can: Search through frequently asked questions (FAQs) Submit a question directly to Technical Support Order Applied Biosystems user documents, MSDSs, certificates of analysis, and other related documents Download PDF documents Obtain information about customer training Download software updates and patches In addition, the Applied Biosystems web site provides a list of telephone and fax numbers that can be used to contact Technical Support for specific products. Applied Biosystems welcomes your comments and suggestions for improving its manuals. You can your comments to: techpubs@appliedbiosystems.com viii July 18, :22 pm, Wallace_GS_howto.fm GPS Explorer Getting Started Guide

10 Introduction to the GPS Explorer Software 1 1 This chapter contains the following sections: Workflow for Identifying Proteins Logging In and Out of GPS Explorer Software GPS Explorer Getting Started Guide 1-1 July 18, :22 pm, Wallace_GS_Ch1.fm

11 Chapter 1 Introduction to the GPS Explorer Software GPS Explorer software retrieves mass spectral data acquired on the Applied Biosystems 4700 Proteomics Analyzer instrument and processes the data for automated identification of proteins. You can create and export reports of the results. Workflow for Identifying Proteins After data is acquired on the 4700 Proteomics Analyzer you: 1. Log in to the GPS Explorer software using the account and password set up by your system administrator. 2. In the GPS Explorer Sample Setup application, prepare a sample set containing spot set data from the 4700 Proteomics Analyzer. 3. Create an analysis (search and selection of job runs) in the sample set. 4. Process the analysis. 5. Analyze the results. 6. Create reports and export results. Experiments Covered in This Guide This guide leads you through four experiments supported by GPS Explorer software (Table 1-1). Table 1-1 GPS Explorer Experiments Covered in this Guide Experiment MS Only for Protein ID (PMF) (Chapter 2) MS/MS Only for Protein ID (Chapter 3) With sample associated by... Well Well or sample set Description Protein identification based on MS mass data of enzymatically-digested proteins. Protein identification based on MS/MS spectral data 1-2 GPS Explorer Getting Started Guide July 18, :22 pm, Wallace_GS_Ch1.fm

12 Workflow for Identifying Proteins Experiment Combined MS and MS/MS Spectra for Protein ID (Chapter 4) Protein ID by Spot Set (MS/MS Spectra Only) (Chapter 5) With sample associated by... Well Sample set Description Two separate protein identifications (one based on MS and the other on MS/MS spectral data) combined for a final protein ID. Protein identification based on MS/MS mass data of a protein mix from an LC separation deposited on a plate GPS Explorer Sample Association Correlation with 4700 Proteomics Analyzer Instrument Data In GPS Explorer software, data from samples spotted on plates can be analyzed in either of two ways: Independently by well Sample Association by Well As a set of samples Sample Association by Sample Set A sample set is a group of well locations and relevant information associated with each location. A sample set is created by the user and stored in the GPS Explorer database. Sample data for the sample set is imported from the 4700 Proteomics Analyzer instrument database from data acquired on the instrument. The correlation between GPS Explorer data groups and 4700 Proteomics Analyzer instrument data groups is indicated in Table 1-2. Note that a sample set can contain one or more spot sets from the 4700 Proteomics Analyzer. Table 1-2 Correlation between GPS Explorer and 4700 Proteomics Analyzer Data Groups. GPS Explorer Data Groups 4700 Proteomics Analyzer Data Groups Project Sample Set Analysis Project Spot Set(s) Run Number GPS Explorer Getting Started Guide 1-3 July 18, :22 pm, Wallace_GS_Ch1.fm

13 Chapter 1 Introduction to the GPS Explorer Software Logging In and Out of GPS Explorer Software Note: Administrators, for information on creating user accounts, see Manage Users on page B-6. To log in to GPS Explorer software using the login name and password created by your system administrator: 1. Click the GPS Explorer desktop icon to display the toolbar. 2. Select in the GPS Explorer toolbar. 3. Enter your User name and Password, then click OK. The GPS Explorer toolbar is activated, making the GPS Explorer applications available for use. Figure 1-1 GPS Explorer Toolbar After you are logged in, the Login button switches to Logout. To log out of GPS Explorer software. 1. Select in the GPS Explorer toolbar. 2. Click Yes at the message prompt. 1-4 GPS Explorer Getting Started Guide July 18, :22 pm, Wallace_GS_Ch1.fm

14 MS Only for Protein ID (PMF) 2 2 This chapter describes how to: Create a Sample Set Create an Analysis Prepare and Submit Samples for Processing Monitor Job Processing View Results GPS Explorer Getting Started Guide 2-1 July 18, :22 pm, Wallace_GS_Ch2.fm

15 Chapter 2 MS Only for Protein ID (PMF) This chapter describes how to use the GPS Explorer software to identify proteins based on MS data of enzymatically digested proteins spotted on a plate. This experiment type is described as MS for protein ID by peptide mass fingerprinting (PMF). About the Example The example used in this chapter is a based on MS data from an alpha amylase sample acquired on an Applied Biosystems 4700 Proteomics Analyzer. Create a Sample Set To create a Sample Set: 1. Click Sample Setup in the GPS Explorer toolbar. 2. In the Project view, select, then right-click Projects. 3. Select New Project, then type a unique name for the new project or accept the default name. 4. Select, then right-click the project in the Project view. 5. Select New Sample Set, then type a unique name for the sample set or accept the default New Sample Set name. 6. In the Sample Set Association box, select By Well in the Association drop-down list for the Alpha Amylase protein identification example. 7. In the Import From area in the center of the screen, click Import Spot Sets. The Import from 4700 Proteomics Analyzer dialog box opens (Figure 2-1 on page 2-3). 2-2 GPS Explorer Getting Started Guide July 18, :22 pm, Wallace_GS_Ch2.fm

16 Create a Sample Set Figure 2-1 Import from 4700 Proteomics Analyzer Dialog Box The left pane lists the 4700 Proteomics Analyzers networked to the GPS Explorer Software and the data available on each system. Note: For information on configuring connections between the GPS Explorer software and the 4700 Proteomics Analyzer computer, see Configure the Instrument on page B In the left pane, click for the appropriate 4700 Proteomics Analyzer to view all the projects in its database. 9. Click next to the appropriate project to view all spot sets in the project. 10. Select the alpha amylase spot set, then click Add to add the spot set to the project. To delete a spot set from the sample set, select the spot set, then click Remove. Note: You cannot delete a spot set from a sample set after it has an analysis associated with it, or after it has been submitted with the sample set for processing. You can repeat this step to add or delete additional spot sets. GPS Explorer Getting Started Guide 2-3 July 18, :22 pm, Wallace_GS_Ch2.fm

17 Chapter 2 MS Only for Protein ID (PMF) 11. Click OK. The selected spot set is imported into the sample set in the GPS Explorer database. Information about the sample set is displayed in the view area. 12. Click Save in the toolbar. Create an Analysis To create an analysis: 1. Select, then right-click the Alpha Amylase sample set in the Project view. 2. Enter a unique name for the analysis, or accept the default name. 3. Click Save to save your changes. 4. In the Analysis By Well section, select MS as the type of analysis (the alpha amylase data set is used for PMF protein ID). 5. Click the Settings bar to open the Analysis Settings dialog box. 6. In the Search Settings tab, select Open Search Settings, then select Alpha Amylase from the list to apply search settings. The search settings are described in Table 2-1. Table 2-1 Search Settings for Alpha Amylase Search Parameters Taxonomy Bacteria (Eubacteria) Setting Report Top... Hits 20 Note: The top hits to report is the maximum number of protein identifications that are returned. The actual number returned can be less because the protein identifications may be below significance before the maximum number of protein identifications is obtained. Database Enzyme SwissProt Trypsin 2-4 GPS Explorer Getting Started Guide July 18, :22 pm, Wallace_GS_Ch2.fm

18 Create an Analysis Search Parameters Setting Max. Missed Cleavages 1 Fixed Modifications Variable Modifications Monoisotopic or Average Peptide Tol Select Oxidation (M) Monoisotopic Note: Because isotopic peaks are resolved, you select Monoisotopic; if they were unresolved, you would select Average. 50 ppm Peptide Charges 1+ Significant Threshold Optional or 85% Note: Significant Threshold is a filter that takes a statistical percent of the Mascot Mowse score (protein score or ion score) greater than the probability that the observed match is a random event and reports, in the Results Browser, the value as a percent confidence value. When a threshold is set, the Results Browser displays only results equal to or above the significant threshold. For example, with the threshold set to 5%, only the top 95% of results that meet the criteria are displayed. Species Information Required Selected. This setting displays the source species information for all identified proteins. Note: Retrieving species information increases database search time. 7. In the MS Peak Filtering tab, select Open Peak Filtering Settings, then select General MS in the list to apply peak filtering settings. the settings are described in Table 2-2 on page 2-6 GPS Explorer Getting Started Guide 2-5 July 18, :22 pm, Wallace_GS_Ch2.fm

19 Chapter 2 MS Only for Protein ID (PMF) Table 2-2 Peak Filter Settings for MS Peak Filter Parameter Setting Mass Range 800 to 4000 Da Minimum S/N 10 Intensity Filter Used Selected Max. Number of Peaks 65 Note: This is the maximum number of peaks submitted for database searching, after all filtering has occurred. Peak Density Used Peak Density Selected 50 per 200 Da Note: This is the number of peaks per 200 Da that is used to create the mass list for database searching. 8. Click Save Settings to apply the settings. 2-6 GPS Explorer Getting Started Guide July 18, :22 pm, Wallace_GS_Ch2.fm

20 Prepare and Submit Samples for Processing Prepare and Submit Samples for Processing To prepare samples for processing: 1. Click Import Job Runs in the Sample Set List section of the main window. The Import From 4700 Proteomics Analyzer window opens (Figure 2-2). Figure 2-2 Import Job Runs Dialog Box 2. In the left pane, click for the appropriate 4700 Proteomics Analyzer to view all the projects in its database. 3. Click to view the lists of analyses for the selected sample set. 4. Select run number 3 from the list of available runs, then click Add. 5. Click OK to import the run. By default, all wells are automatically selected for submission. GPS Explorer Getting Started Guide 2-7 July 18, :22 pm, Wallace_GS_Ch2.fm

21 Chapter 2 MS Only for Protein ID (PMF) 6. To select specific samples for processing, do one of the following: Right-click the well grid, then select Remove Samples. In the Submit column of the sample Information table, select the check boxes that correspond to the samples of interest (Figure 2-3). When you are done, right-click the well grid, then select Select To Submit. Figure 2-3 Selection of Samples in Sample Information Table Right-click the well grid, then select Remove Samples. Right-click the well grid, select Group Selection or Single Selection, then select the wells of interest (Figure 2-4 on page 2-9). When you are done, right-click the well grid, then select Select To Submit. 2-8 GPS Explorer Getting Started Guide July 18, :22 pm, Wallace_GS_Ch2.fm

22 Prepare and Submit Samples for Processing Figure 2-4 Selection of Samples in the Plate View 7. Click Save, to save your analysis. 8. Click in the toolbar to submit the samples for processing. Note: The server must be running before you submit the samples for processing. Otherwise the analysis remains in the waiting state. See Manage Database Functions on page B-5. GPS Explorer Getting Started Guide 2-9 July 18, :22 pm, Wallace_GS_Ch2.fm

23 Chapter 2 MS Only for Protein ID (PMF) Monitor Job Processing To monitor the progress of your job: 1. Click in the GPS Explorer toolbar. The Project view lists the projects with sample sets and analyses that are ready for processing. The Job list shows the job queue (Figure 2-5). (Jobs that have already been processed or are not yet ready for processing are not listed in the job queue.) Figure 2-5 Job Manager Project View and Job List In addition, the right side of the screen displays information on the selected analysis. For association by well, a plate view shows job status and legend. 2. Navigate to the analysis you created earlier in this chapter by clicking in the Project view next to the sample set created in the previous chapter. The analysis you submitted is displayed in the sample set under the project. Note: You can also select and right-click an analysis node in the Project view, then select Submit to Processing Queue GPS Explorer Getting Started Guide July 18, :22 pm, Wallace_GS_Ch2.fm

24 Monitor Job Processing 3. In the Job Status view area, information for the job is listed in the queue (Figure 2-6). The list displays the order in which jobs will be processed. Figure 2-6 Job Manager Job List Status Display Note: If the server is shut down while processing a job, the job status is suspended. Processing does not restart until the server is running again. 4. To pause a job, when the job status is Active, select Job Queue > Pause. The job remains in the processing list in a waiting state. 5. To restart a paused job, select the job in the queue, then select Job Queue > Restart. The job status changes to and remains Active while the job is processing. If the job fails, it remains in the queue with a job status of Failed. You can check the Event Log to determine the cause of the error (see step 7 on page 2-12). 6. With the job status Active, refer to the Plate view on the right of the screen. The colors indicate the progress of the processing (Figure 2-7 on page 2-12). GPS Explorer Getting Started Guide 2-11 July 18, :22 pm, Wallace_GS_Ch2.fm

25 Chapter 2 MS Only for Protein ID (PMF) Figure 2-7 Job Manager Plate View and Legend 7. Right-click the GPS Explorer toolbar, then select Event Log to view system information related to your job (Figure 2-8). Figure 2-8 Event Log After the job has finished processing, it is removed from the queue GPS Explorer Getting Started Guide July 18, :22 pm, Wallace_GS_Ch2.fm

26 View Results View Results To view the results of the MS analysis: 1. Click in the GPS Explorer toolbar. Note: You can open multiple Results Browser windows at one time. See Configure the Desktop on page B-2 for more information. 2. In the Project view, navigate to the analysis you submitted for processing, then do one of the following: Click the link show the results. Right-click the Project view, then select Open. Double-click the analysis in the Project view. 3. Right-click the Plates tab, then select Well Color Legend to view what each well color indicates. 4. To set the Plate view settings, select View > Plate > Settings. The Plate Settings dialog box opens(figure 2-9 on page 2-14). 5. Select the Protein Identification tab to view the confidence settings (inner well colors) of the well representations. Note: For information on the Confidence Interval, see the GPS Explorer Software Online Help. Three confidence levels are provided, and color settings for each can be customized (Figure 2-9 on page 2-14). GPS Explorer Getting Started Guide 2-13 July 18, :22 pm, Wallace_GS_Ch2.fm

27 Chapter 2 MS Only for Protein ID (PMF) b Figure 2-9 Plate Settings Dialog Box 6. In the following fields, type the indicated values. High Confidence: Greater Than Or Equal To 97%. Low Confidence: 90 to 97%. No Confidence: Less Than Or Equal To 90%. Note: Colors identifying high, low, and no confidence are determined by the percentage values set in the Confidence% input boxes. The Confidence% corresponds to the Protein confidence interval (c.i.) for MS spectra and the Total Ion Score confidence interval (c.i.) for MS/MS spectra in the table view. 7. Click Apply to apply the changes. 8. Click OK to save and view your changes to the Plate view. 9. In the Plate view, select well 11. Results for well 11 are displayed in the Proteins In Current Well table and in the spectrum (Figure 2-10 on page 2-15) GPS Explorer Getting Started Guide July 18, :22 pm, Wallace_GS_Ch2.fm

28 View Results Figure 2-10 Results Browser Display of Results by Well Note: Your protein identifications may vary depending on which version of the sequence database you are searching against. 10. Select then right-click the Metric Plot tab, then select Settings. The Metric Table tab displays the same data as the Metric Plot tab, but in table format. 11. Select settings for the Metric Plot parameters as indicated in Table 2-3 on page GPS Explorer Getting Started Guide 2-15 July 18, :22 pm, Wallace_GS_Ch2.fm

29 Chapter 2 MS Only for Protein ID (PMF) Table 2-3 Metric Plot Parameters Parameter Available Settings (one per parameter) Plate Display One plate All plates Trace Style Point Connected Stick X-axis Well index Well order (if you specify well order in sample setup) Y-axis Score Protein database search score. Raw Reconstructed Ion Current Calculated by summing all mass intensities (peak areas) of the MS spectrum associated with each well. Submitted Reconstructed Ion Current Calculated by summing all filtered mass intensities (peak areas) for each well spot submitted for database searching 12. In the toolbar, click Plate, then click Spectrum to hide the Plate and Spectrum views and expand the Protein Summary table. Note: The Protein Summary table lists all proteins on a plate and the corresponding peptides returned from the database search. Proteins containing the same peptide are grouped together GPS Explorer Getting Started Guide July 18, :22 pm, Wallace_GS_Ch2.fm

30 View Results Click + to the left of the first item, Alpha-Amylase, in the Protein In Current Well table to display the peptide results (Figure 2-11). Figure 2-11 Peptide Results 13. Click Spectrum in the toolbar to redisplay the spectrum. 14. With well 11 selected in the Plate view, drag across the spectrum peaks between 1300 and 1500 Da. To zoom in on this mass range (Figure 2-12 on page 2-18). GPS Explorer Getting Started Guide 2-17 July 18, :22 pm, Wallace_GS_Ch2.fm

31 Chapter 2 MS Only for Protein ID (PMF) Figure 2-12 Zoomed Spectrum Right-click to zoom out of the spectrum. 15. Select the Peaks tab to display the Peak list. 16. To view the Peak Color Legend, right-click the Spectrum tab, then select Peak Color Legend. The legend box shows color representations (Figure 2-13). Figure 2-13 Peak List Table with Legend 17. Right-click the Peak list, select Select All, then right-click and select Copy GPS Explorer Getting Started Guide July 18, :22 pm, Wallace_GS_Ch2.fm

32 View Results 18. Open Windows Notepad, then select Edit > Paste. The peak list is pasted into Notepad (Figure 2-14). Figure 2-14 Peak List Copied to Notepad 19. Save and close the Notepad file. You can edit the file later to fix column alignment or add or delete data before importing the contents into a spreadsheet. 20. To view the HTML file created by the Mascot software, in the Plates tab select and right-click a spot, then select Open DB Results to view the Mascot results returned from the database search. For more information on using the Results Browser, see the GPS Explorer Software Online Help. For information on creating reports from your data, see Chapter 6, Creating Reports. GPS Explorer Getting Started Guide 2-19 July 18, :22 pm, Wallace_GS_Ch2.fm

33 Chapter 2 MS Only for Protein ID (PMF) 2-20 GPS Explorer Getting Started Guide July 18, :22 pm, Wallace_GS_Ch2.fm

34 MS/MS Only for Protein ID 3 3 This chapter describes how to: Create a Sample Set Create an Analysis Prepare and Submit Samples for Processing Monitor Job Processing View Results GPS Explorer Getting Started Guide 3-1 July 18, :22 pm, Wallace_GS_Ch3.fm

35 Chapter 3 MS/MS Only for Protein ID This chapter describes how to use GPS Explorer Software to identify proteins based on MS/MS mass data of enzymatically-digested proteins spotted on a plate. About the Example The example used in this chapter is a based on MS/MS spectral data from a Beta Galactosidase sample acquired on an Applied Biosystems 4700 Proteomics Analyzer. Create a Sample Set To create a Sample Set: 1. Click Sample Setup in the GPS Explorer software toolbar. 2. In the Project view in the main screen, select, then right-click Projects. 3. Select New Project, then type a unique name for the new project, or accept the default name. 4. Select, then right-click the new project in the Project view. 5. Select New Sample Set, then type a unique name for the sample set or accept the default New Sample Set name. 6. In the Sample Set Association box, select By Well in the Association drop-down list. 7. In the Import From area in the center of the screen, click Import Spot Sets. The Import from 4700 Proteomics Analyzer dialog box opens. The left pane lists the 4700 Proteomics Analyzers networked to the GPS Explorer Software and the data available on each system. 8. In the left pane, click for the appropriate 4700 Proteomics Analyzer to view all the projects in its database. 9. Click next to the appropriate project to view all spot sets in the project. 3-2 GPS Explorer Getting Started Guide July 18, :22 pm, Wallace_GS_Ch3.fm

36 Create a Sample Set 10. Select a spot set with acquired beta galactosidase data, then click Add to add the spot set to the project (Figure 3-1). Figure 3-1 Import from 4700 Proteomics Analyzer Dialog Box To delete a selected spot set from the sample set, click Remove. Note: You cannot delete a spot set from a sample set after it has an analysis associated with it or after it has been submitted with the sample set for processing. 11. Repeat step 10 to add or delete additional spot sets. 12. Click OK. The selected spot sets are imported into the sample set in the GPS Explorer Software database. Information about the sample set is displayed in the data area. 13. Click Save in the toolbar. GPS Explorer Getting Started Guide 3-3 July 18, :22 pm, Wallace_GS_Ch3.fm

37 Chapter 3 MS/MS Only for Protein ID Create an Analysis To create an Analysis: 1. Select, then right-click the Beta Galactosidase sample set in the Project view. 2. Enter a unique name for the analysis, or accept the default name. 3. Click Save to save your changes. 4. In the Analysis By Well section, select MS/MS as the type of analysis (the beta galactosidase data set is used in this experiment). 5. Click the Settings bar to open the Analysis Settings dialog box. 6. In the Search Settings tab, select Open Search Settings, then select Beta Galactosidase from the list to apply search settings. Search settings are described in Table 3-1. Table 3-1 Search Settings for Beta Galactosidase Search Parameter Setting Taxonomy Escherichia coli Report Top... Hits 20 Note: The top hits to report is the maximum number of protein identifications that are returned. The actual number returned can be less than the maximum number because the protein identifications may be below significance before the maximum number of protein identifications is obtained. Database Enzyme SwissProt Trypsin Max. Missed Cleavages 1 Fixed Modifications Variable Modifications Selected Oxidation (M) 3-4 GPS Explorer Getting Started Guide July 18, :22 pm, Wallace_GS_Ch3.fm

38 Create an Analysis Search Parameter Monoisotopic or Average Peptide Tol Monoisotopic Note: Because isotopic peaks are resolved, you select Monoisotopic; if they were unresolved, you would select Average. 75 ppm Setting Peptide Charges 1+ Significant Threshold Optional or 85% Note: Significant Threshold is a filter that takes a statistical percent of the Mascot Mowse score (protein score or ion score) greater than the probability that the observed match is a random event and reports, in the Results Browser, the value as a percent confidence value. When a threshold is set, the Results Browser displays only results equal to or above the significant threshold. For example, with the threshold set to 5%, only the top 95% of results that meet the criteria are displayed. Species Information Required MS/MS Tol Selected. This setting displays the source species information for all identified proteins. Note: Retrieving species information increases database search time. 0.4 Da 7. In the MS/MS Peak Filtering tab, select Open Peak Filtering Settings, then select General MS-MS from the list to apply peak filtering settings. The settings are described in Table 3-2. Table 3-2 Peak Filter Settings for MS/MS Peak Filter Parameter Mass Range Setting 60 to 20 Da below the precursor mass Minimum S/N 10 Intensity Filter Used Selected GPS Explorer Getting Started Guide 3-5 July 18, :22 pm, Wallace_GS_Ch3.fm

39 Chapter 3 MS/MS Only for Protein ID Peak Filter Parameter Setting Max. Number of Peaks 65 Note: This is the maximum number of peaks submitted for database searching, after all filtering has occurred. Peak Density Used Peak Density Selected 50 per 200 Da Note: This is the number of peaks per 200 Da that are used to create the mass list for database searching. 8. Click Save Settings to apply the settings. 9. When queried to skip base MS peak filtering settings, click Yes. 3-6 GPS Explorer Getting Started Guide July 18, :22 pm, Wallace_GS_Ch3.fm

40 Prepare and Submit Samples for Processing Prepare and Submit Samples for Processing To prepare and submit samples for processing: 1. Click Import Job Runs in the Sample Set List section of the main window. The Import From 4700 Proteomics Analyzer window opens (Figure 3-2). Figure 3-2 Importing Job Runs Note: You can import base MS analyses with the MS/MS runs. However, the base MS data are used only with combined MS and MS/MS analyses. 2. In the left pane, click for the appropriate 4700 Proteomics Analyzer to view all the projects in its database. 3. Click to view the lists of analyses for the selected sample set. 4. Select the run from the list of available runs, then click Add. GPS Explorer Getting Started Guide 3-7 July 18, :22 pm, Wallace_GS_Ch3.fm

41 Chapter 3 MS/MS Only for Protein ID 5. Click OK, to import the run. By default, all wells in a sample set are selected for processing. 6. To select samples for processing, do one of the following: Right-click the well grid, then select Remove Samples. In the Submit column of the Sample Information table, select the check boxes that correspond to the samples of interest (Figure 3-3). When you are done, right-click the well grid, then select Select To Submit. Figure 3-3 Selection of Samples from Sample Information Table Right-click the well grid, then select Remove Samples. Right-click the well grid, select Group Selection or Single Selection, then select the wells of interest (Figure 3-4 on page 3-9). When you are done, right-click the well grid, then select Select To Submit. 3-8 GPS Explorer Getting Started Guide July 18, :22 pm, Wallace_GS_Ch3.fm

42 Prepare and Submit Samples for Processing Figure 3-4 Selection of Samples in the Plate View 7. Click Save, to save your analysis. 8. Click in the toolbar to submit the samples for processing. Note: The server must be running before you submit the samples. Otherwise, the analysis remains in the waiting state. See Manage Database Functions on page B-5. GPS Explorer Getting Started Guide 3-9 July 18, :22 pm, Wallace_GS_Ch3.fm

43 Chapter 3 MS/MS Only for Protein ID Monitor Job Processing To monitor the progress of your job: 1. Click in the GPS Explorer toolbar. The Project view lists projects with sample sets and analyses that are ready for processing. The Job list shows the job queue (Figure 3-5). (Jobs that have already been processed or are not yet ready for processing are not listed). Figure 3-5 Job Manager Project View and Job List In addition, the right of the screen displays information on the selected analysis. For association by well, a Plate view shows job status and legend. 2. In the Project view, navigate to the analysis you created earlier in this chapter by clicking in the Project view next to the sample set created in the previous chapter. The analysis you submitted is displayed in the sample set under the project. 3. In the Job Status view, information for the job is listed in the queue (Figure 3-6). The list displays the order in which jobs will be processed. Figure 3-6 Job Manager Job List Status Display 3-10 GPS Explorer Getting Started Guide July 18, :22 pm, Wallace_GS_Ch3.fm

44 Monitor Job Processing Note: If the server is shut down while processing a job, the job status is suspended. Processing does not restart until the server is running again. 4. To pause a job, when the job status is Active, select the job in the queue, then select Job Queue > Pause. The job remains in the processing list in a waiting state. 5. To restart a paused job, select the job in the queue, then select Job Queue > Restart. The status changes to and remains Active while the job is processing. If the job fails, it remains in the queue with a job status of Failed. 6. With the job status Active, refer to the Plate view on the right of the screen. The colors indicate the progress of the processing (Figure 3-7). Figure 3-7 Job Manager Plate View and Legend 7. Right-click the GPS Explorer toolbar, then select Event Log to view system information related to the job (Figure 3-8 on page 3-12). GPS Explorer Getting Started Guide 3-11 July 18, :22 pm, Wallace_GS_Ch3.fm

45 Chapter 3 MS/MS Only for Protein ID Figure 3-8 Event Log After the job has finished processing, it is removed from the queue GPS Explorer Getting Started Guide July 18, :22 pm, Wallace_GS_Ch3.fm

46 View Results View Results To view the results of the MS/MS analysis: 1. Click in the GPS Explorer toolbar. Note: You can open multiple Results Browser windows at one time. See Configure the Desktop on page B-2 for more information. 2. In the Project view, navigate to the analysis you submitted for processing, then do one of the following: Click the link show the results. Right-click the Project view, then select Open. Double-click the analysis in the Project view. 3. Right-click the Plates tab, then select Well Color Legend to view what each color indicates. Note: For information on the Confidence Interval, see the GPS Explorer Software Online Help. 4. To set the Plate view settings, select View > Plate > Settings. The Plate Settings dialog box opens. 5. Select the Protein Identification tab to view the confidence settings (inner well colors) of the well representations. The confidence settings are based on the Significant Score calculated from the database search results. Three confidence levels are provided, and color settings can be customized (Figure 3-9 on page 3-14). GPS Explorer Getting Started Guide 3-13 July 18, :22 pm, Wallace_GS_Ch3.fm

47 Chapter 3 MS/MS Only for Protein ID Figure 3-9 Plate Settings Dialog Box 6. In the following fields, type the indicated values. High Confidence: Greater Than or Equal to 97%. Low Confidence: 90 to 97%. No Confidence: Less Than or Equal to 90%. Note: High, low, and no confidence colors are determined by percentage values in the Confidence% input boxes in the Protein Identification tab. The Confidence% corresponds to the Protein confidence interval (c.i.) for MS spectra and the Total Ion Score confidence interval (c.i.) for MS/MS spectra in the Table view. 7. Click Apply to apply the settings. Note: For information on customizing the well grid display of confidence settings in the well grid, see Customize Well-Color Display on page A-5 8. Click OK to save and view your changes in the Plate view. 9. In the Plate view, select well 1. Results for well 1 are displayed. Note: Your protein identifications may vary depending on which version of the sequence database you are searching against GPS Explorer Getting Started Guide July 18, :22 pm, Wallace_GS_Ch3.fm

48 View Results 10. Select and right-click the Metric Plot tab, then select settings (Figure 3-10). Figure 3-10 Results Browser Display of Metric Plot Note: The Protein Summary table lists all proteins on the plate and the corresponding peptides returned from the database search. Proteins containing the same peptide are grouped together. 11. Select settings for the Metric Plot parameters as indicated in Table 3-3 on page GPS Explorer Getting Started Guide 3-15 July 18, :22 pm, Wallace_GS_Ch3.fm

49 Chapter 3 MS/MS Only for Protein ID Table 3-3 Metric Plot Parameters Parameter Available Settings (one per parameter) Plate Display One plate All plates Trace Style Point Connected Stick X-axis Well index Well order Y-axis Score Protein database search score. Raw Reconstructed Ion Current Calculated by summing all mass intensities (peak areas) of the MS spectrum associated with each well. Submitted Reconstructed Ion Current Calculated by summing all filtered mass intensities (peak areas) for each well spot submitted for the database search. 12. In the Protein Results table, click + to the left of the first item, Beta Galactactosidase, in the Proteins In Current Well table to display peptide results (Figure 3-11 on page 3-17). Note: Clicking a peptide updates the Spectrum view GPS Explorer Getting Started Guide July 18, :22 pm, Wallace_GS_Ch3.fm

50 View Results Figure 3-11 Peptide Results 13. In the Spectrum tab, drag across the spectrum peaks below 200 Da to zoom in on the selected peaks (Figure 3-12). Figure 3-12 Zoomed Spectrum Right-click to zoom out of the spectrum. 14. Select the Peaks tab to display the Peak list. 15. To view the Peak Color Legend, right-click the Spectrum tab, then select Peak Color Legend. GPS Explorer Getting Started Guide 3-17 July 18, :22 pm, Wallace_GS_Ch3.fm

51 Chapter 3 MS/MS Only for Protein ID The legend box shows color representations (Figure 3-13). Figure 3-13 Peak List Table with Legend 16. Right-click the Peak list, select Select All, then right-click and select Copy. 17. Open Windows Notepad, then select Edit > Paste. The Peak list is pasted into Notepad (Figure 3-14 on page 3-19) GPS Explorer Getting Started Guide July 18, :22 pm, Wallace_GS_Ch3.fm

52 View Results Figure 3-14 Peak List Copied to Notepad 18. Save and close the Notepad file. You can edit the file later to fix column alignment or add or delete data before importing the contents into a spreadsheet. 19. To view the HTML file created by the Mascot software, select the Plates tab. 20. Select and right-click a spot, then select Open DB Results to view the Mascot results returned from the database search. For more information on using the Results Browser, see the GPS Explorer Software Online Help. For information on creating reports from your data, see Chapter 6, Creating Reports. GPS Explorer Getting Started Guide 3-19 July 18, :22 pm, Wallace_GS_Ch3.fm

53 Chapter 3 MS/MS Only for Protein ID 3-20 GPS Explorer Getting Started Guide July 18, :22 pm, Wallace_GS_Ch3.fm

54 Combining MS and MS/MS Spectra for Protein ID 4 4 This chapter describes how to: Create a Sample Set Create an Analysis Prepare and Submit Samples for Processing Monitor Job Processing View Results GPS Explorer Getting Started Guide 4-1 July 18, :22 pm, Wallace_GS_Ch4.fm

55 Chapter 4 Combining MS and MS/MS Spectra for Protein ID This chapter describes how to use the GPS Explorer software to identify proteins based on combined MS-MS/MS mass data of enzymatically-digested proteins spotted on a plate. The GPS Explorer software performs two separate protein identification searches, one based on PMF and the other on MS/MS spectral data. Results are combined for a final protein ID. About the Example The example used in this chapter is based on MS-MS/MS spectral data from a Beta Galactosidase sample acquired on an Applied Biosystems 4700 Proteomics Analyzer. Create a Sample Set To create a sample set: 1. Click Sample Setup in the GPS Explorer toolbar. 2. In the Project view, select then right-click Projects. 3. Select New Project, then type a unique name for the new project, or accept the default name. 4. Select, then right-click the project in the Project view. 5. Select New Sample Set, then type a unique name for the sample set, or accept the default New Sample Set name. 6. In the Sample Set Association box, select By Well in the Association drop-down list. 7. In the Import From area in the center of the screen, click Import Spot Sets. The Import from 4700 Proteomics Analyzer dialog box opens. The left pane lists the 4700 Proteomics Analyzers networked to the GPS Explorer software and the data available on each system. 8. In the left pane, click for the appropriate 4700 Proteomics Analyzer to view all the projects in its database. 9. Click next to the appropriate project to view all spot sets in the project. 4-2 GPS Explorer Getting Started Guide July 18, :22 pm, Wallace_GS_Ch4.fm

56 Create a Sample Set 10. Select a spot set with acquired beta galactosidase data, then click Add to add the spot set to the project (Figure 4-1). Figure 4-1 Import from 4700 Proteomics Analyzer Dialog Box To delete a selected spot set from the sample set, click Remove. Note: You cannot delete a spot set from a sample set after it has an analysis associated with it or after it has been submitted with the sample set for processing. 11. Repeat step 10 to add or delete additional spot sets. 12. Click OK. The selected spot sets are imported into the sample set in the GPS Explorer database. Information about the sample set is displayed in the view area. Note: For information on displaying user-specified columns in the sample information table, see Display User-Defined Sample Information on page A Click Save in the toolbar. GPS Explorer Getting Started Guide 4-3 July 18, :22 pm, Wallace_GS_Ch4.fm

57 Chapter 4 Combining MS and MS/MS Spectra for Protein ID Create an Analysis To create an analysis: 1. Select, then right-click the Beta Galactosidase sample set in the Project view. 2. Enter a unique name for the analysis, or accept the default name. 3. Click Save to save your changes. 4. In the Analysis By Well section, select MS/MS as the type of analysis (beta galactosidase is used in this experiment), then select the Combined check box (Figure 4-2). Figure 4-2 Specifying a Combined MS and MS/MS Analysis 5. Click the Settings bar to open the Analysis Settings dialog box. 6. In the Search Settings tab, select Open Search Settings, then select Beta Galactosidase from the list to apply search settings. The search settings are described in Table 4-1. Table 4-1 Search Settings for Beta Galactosidase Parameter Setting Taxonomy Escherichia coli Report Top... Hits 20 Note: The top hits to report is the maximum number of protein identifications that are returned. The actual number returned can be less because the protein identifications may be below significance before the maximum number of protein identifications is obtained. Database SwissProt 4-4 GPS Explorer Getting Started Guide July 18, :22 pm, Wallace_GS_Ch4.fm

58 Create an Analysis Parameter Setting Enzyme Trypsin Max. Missed Cleavages 1 Fixed Modifications Variable Modifications Monoisotopic or Average Peptide Tol None Oxidation (M) Monoisotopic Note: Because isotopic peaks are resolved, you select Monoisotopic; if they were unresolved, you would select Average. 75 ppm Peptide Charges 1+ Significant Threshold Optional or 85% Note: Significant Threshold is a filter that takes a statistical percent of the Mascot Mowse score (protein score or ion score) greater than the probability that the observed match is a random event and reports, in the Results Browser, the value as a percent confidence value. When a threshold is set, the Results Browser displays only results equal to or above the significant threshold. For example, with the threshold set to 5%, only the top 95% of results that meet the criteria are displayed. Species Information Required MS/MS Tol Selected. This setting displays the species information for all identified proteins. Note: Retrieving species information increases database search time. 0.4 Da 7. Click Save, then enter a unique file name to save the search settings for later use. GPS Explorer Getting Started Guide 4-5 July 18, :22 pm, Wallace_GS_Ch4.fm

59 Chapter 4 Combining MS and MS/MS Spectra for Protein ID 8. In the MS/MS Peak Filtering tab, select Open Peak Filtering Settings, then select General MS-MS from the list to apply peak filtering settings. The settings are described in Table 4-2. Table 4-2 Peak Filter Settings for MS/MS Mass Range Parameter Setting 60 to 20 Da below the precursor mass Minimum S/N 10 Intensity Filter Used Selected Max. Number of Peaks 65 Note: This is the maximum number of peaks submitted for database searching, after all filtering has occurred. Peak Density Used Peak Density: Selected 50 per 200 Da Note: This is the number of peaks per 200 Da that is used to create the mass list for database searching. 9. In the Base MS Peak Filtering tab, select Open Peak Filtering Settings, then select General MS from the list to apply peak filtering settings. The settings are described in Table 4-3. Table 4-3 Base Peak Filter Settings for MS Parameter Setting Mass Range 800 to 4000 Da Minimum S/N 10 Intensity Filter Used Selected Max. Number of Peaks 50 Note: This is the maximum number of peaks submitted for database searching, after all filtering has occurred. Peak Density Used Selected 4-6 GPS Explorer Getting Started Guide July 18, :22 pm, Wallace_GS_Ch4.fm

60 Create an Analysis Parameter Peak Density 50 per 200 Da Setting Note: This is the number of peaks per 200 Da that is used to create the mass list for database searching. 10. Click Save Settings to apply the settings. GPS Explorer Getting Started Guide 4-7 July 18, :22 pm, Wallace_GS_Ch4.fm

61 Chapter 4 Combining MS and MS/MS Spectra for Protein ID Prepare and Submit Samples for Processing To prepare and submit samples for processing: 1. Click Import Job Runs in the Sample List section of the main window. The Import From 4700 Proteomics Analyzer window opens (Figure 4-3). Figure 4-3 Importing Job Runs 2. In the left pane, click for the appropriate 4700 Proteomics Analyzer to view all the projects in its database. 3. Click to expand the lists of analyses for the selected sample set. 4. Select the run from the list of available runs, then click Add. 5. Click OK to import the run. 4-8 GPS Explorer Getting Started Guide July 18, :22 pm, Wallace_GS_Ch4.fm

62 Prepare and Submit Samples for Processing 6. To select samples for processing, do one of the following: Right-click the well grid, then select Remove Samples. In the Submit column of the Sample Information table, select the check boxes that correspond to the samples of interest (Figure 4-4). When you are done, right-click the well grid, then select Select To Submit. Figure 4-4 Selection of Samples from Sample Information Table Right-click the well grid, then select Remove Samples. Right-click the well grid, select Group Selection or Single Selection, then select the wells of interest (Figure 4-5 on page 4-10). When you are done, right-click the well grid, then select Select To Submit. GPS Explorer Getting Started Guide 4-9 July 18, :22 pm, Wallace_GS_Ch4.fm

63 Chapter 4 Combining MS and MS/MS Spectra for Protein ID Figure 4-5 Selection of Samples in the Plate View 7. Click Save to save your analysis. 8. Click in the toolbar to submit the samples for processing. Note: The server must be running before you submit the analysis. Otherwise, the analysis remains in the waiting state. See Manage Database Functions on page B GPS Explorer Getting Started Guide July 18, :22 pm, Wallace_GS_Ch4.fm

64 Monitor Job Processing Monitor Job Processing To monitor the progress of your job processing: 1. Click in the GPS Explorer toolbar. The Project view lists projects with sample sets and analyses that are ready for processing. The Job list shows the job queue (Figure 4-6). (Jobs that have been processed or are not yet ready for processing are not listed). Figure 4-6 Job Manager Project View and Job List In addition, the right of the screen displays information on the selected analysis. For association by well, a Plate view shows job status and legend. 2. In the Project view, navigate to the analysis you created earlier in this chapter by clicking in the Project view next to the sample set created in the previous chapter. The analysis you submitted is displayed in the sample set under the project. 3. In the Job Status view area, information for the job is listed in the queue (Figure 4-7). The list displays the order in which jobs are processed. Figure 4-7 Job Manager Job List Status Display GPS Explorer Getting Started Guide 4-11 July 18, :22 pm, Wallace_GS_Ch4.fm

65 Chapter 4 Combining MS and MS/MS Spectra for Protein ID Note: If the server is shut down while processing a job, the job status is suspended. Processing does not restart until the server is running again. 4. To pause a job, when the job status is Active, select the job in the queue, then select Job Queue > Pause. The job remains in the processing list in a waiting state. 5. To restart a paused job, select the job in the queue, then select Job Queue > Restart. The status changes to and remains Active while the job is processing. If the job fails, it remains in the queue with a job status of Failed. 6. With the job status Active, refer to the Plate view on the right of the screen. The colors indicate the progress of the processing (Figure 4-8). Figure 4-8 Job Manager Plate View and Legend 4-12 GPS Explorer Getting Started Guide July 18, :22 pm, Wallace_GS_Ch4.fm

66 Monitor Job Processing 7. Right-click the GPS Explorer toolbar, then select Event Log to view system information related to the job (Figure 4-9). Figure 4-9 Event Log After the job has finished processing, it is removed from the queue. GPS Explorer Getting Started Guide 4-13 July 18, :22 pm, Wallace_GS_Ch4.fm

67 Chapter 4 Combining MS and MS/MS Spectra for Protein ID View Results To view results of the MS-MS/MS combined analysis: 1. Click in the GPS Explorer toolbar. 2. In the Project view, navigate to the analysis you submitted for processing, then do one of the following: Click the link show the results. Right-click the Project view, then select Open. Double-click the analysis in the Project view. 3. Right-click the Plates tab, then select Well Color Legend to view what each color indicates. 4. To set the Plate view settings, select View > Plate > Settings. The Plate Settings dialog box opens. 5. Select the Protein Identification tab to view the confidence settings (inner well colors) of the well representations. The confidence settings are based on the Significant Score calculated from the database search results. Three confidence levels are provided and color settings for each can be customized (Figure 4-10 on page 4-15) GPS Explorer Getting Started Guide July 18, :22 pm, Wallace_GS_Ch4.fm

68 View Results Figure 4-10 Plate Settings Dialog Box 6. In the following fields, type the indicated values. High Confidence: Greater Than Or Equal To 97%. Low Confidence: 90 to 97%. No Confidence: Less Than Or Equal To 90%. 7. Click Apply to apply the changes Note: For information on the Confidence Interval, see the GPS Explorer Software Online Help. 8. Click OK to save and view your changes in the Plate view. 9. In the Plate view, select well 16. Results for well 16 are displayed in the Proteins table and in the spectrum (Figure 4-11 on page 4-16). GPS Explorer Getting Started Guide 4-15 July 18, :22 pm, Wallace_GS_Ch4.fm

69 Chapter 4 Combining MS and MS/MS Spectra for Protein ID Figure 4-11 Results Browser Display of Results by Well Note: Your protein identifications may vary depending on which version of the sequence database you are searching against. 10. Select and right-click the Metric Plot tab, then select Settings. 11. Select settings for Metric Plot parameters as indicated in Table 4-4 on page GPS Explorer Getting Started Guide July 18, :22 pm, Wallace_GS_Ch4.fm

70 View Results Table 4-4 Metric Plot Parameters Parameter Available Settings (one per parameter) Plate Display One plate All plates Trace Style Point Connected Stick X-axis Well index Well order Y-axis Score Protein database search score. Raw Reconstructed Ion Current Calculated by summing all mass intensities (peak areas) of the MS spectrum associated with each well. Submitted Reconstructed Ion Current Calculated by summing all filtered mass intensities (peak areas) for each well spot submitted for the database search 12. In the toolbar, click Plate, then click Spectrum to hide the Plate and Spectrum views and expand the Protein table. The Protein Summary table lists all proteins and corresponding peptides returned from database search. Proteins containing the same peptide are grouped together. GPS Explorer Getting Started Guide 4-17 July 18, :22 pm, Wallace_GS_Ch4.fm

71 Chapter 4 Combining MS and MS/MS Spectra for Protein ID 13. Click + to the left of the first item, Beta Galactosidase, to display the Peptides table for that protein (Figure 4-12). Figure 4-12 Peptide Results Table 14. Click Spectrum in the toolbar to redisplay the spectrum. 15. With well 16 selected in Plate view, drag across the spectrum peaks between 400 and 750 Da. The display zooms in on the selected peaks (Figure 4-13 on page 4-19) GPS Explorer Getting Started Guide July 18, :22 pm, Wallace_GS_Ch4.fm

72 View Results Figure 4-13 Zoomed Spectrum Right-click to zoom out of the spectrum. 16. Click the Peaks tab to display the Peak list. 17. To view the Peak Color Legend, right-click the Spectrum tab, then select Peak Color Legend. Figure 4-14 Peak List Table with Legend 18. Right-click the Peak list, select Select All, then right-click and select Copy. 19. Open Windows Notepad, then select Edit > Paste. The Peak list is pasted into Notepad (Figure 4-15 on page 4-20). GPS Explorer Getting Started Guide 4-19 July 18, :22 pm, Wallace_GS_Ch4.fm

73 Chapter 4 Combining MS and MS/MS Spectra for Protein ID Figure 4-15 Peak List Copied to Notepad 20. Save and close the Notepad file. You can edit the file later to fix column alignment or add or delete data before importing the contents into a spreadsheet. 21. To view the HTML file created by the Mascot software, select then right-click the Plates tab. 22. Select and right-click a spot, then select Open DB Results to view the Mascot results returned from the database search. For more information on using the Results Browser, see the GPS Explorer Software Online Help. For information on creating reports from your data, see Chapter 6, Creating Reports GPS Explorer Getting Started Guide July 18, :22 pm, Wallace_GS_Ch4.fm

74 Protein ID by Sample Set (MS/MS Spectra Only) 5 5 This chapter describes how to: Create a Sample Set Create an Analysis Prepare and Submit Samples for Processing Monitor Job Processing View Results GPS Explorer Getting Started Guide 5-1 July 18, :22 pm, Wallace_GS_Ch5.fm

75 Chapter 5 Protein ID by Sample Set (MS/MS Spectra Only) This chapter describes how to use the GPS Explorer software to identify proteins based on MS/MS mass spectral data of a protein mix from an LC separation spotted on a plate. About the Example The example used in this chapter is based on an LC-fractionated protein mix acquired on the Applied Biosystems 4700 Proteomics Analyzer. The LC eluent was tracked in rows across a MALDI plate, and data were acquired along the tracks. Create a Sample Set To create a sample set: 1. Click Sample Setup in the GPS Explorer toolbar. 2. In the Project view in the main screen, select, then right-click Projects. 3. Select New Project, then type a unique name for the new project, or accept the default name. 4. Select, then right-click the project in the Project view. 5. Select New Sample Set, then type a unique name for the sample set, or accept the default New Sample Set name. 6. In the Sample Set Association box, select By Sample Set. 7. In the Import From area in the center of the screen, click Import Spot Sets. The Import from 4700 Proteomics Analyzer dialog box opens. The left pane lists the 4700 Proteomics Analyzers networked to the GPS Explorer software and the data available on each system. 8. In the left pane, click for the appropriate 4700 Proteomics Analyzer to view all the projects in its database. 9. Click next to the appropriate project to view all spot sets in the project. 5-2 GPS Explorer Getting Started Guide July 18, :22 pm, Wallace_GS_Ch5.fm

76 Create a Sample Set 10. Select the 9 protein mix spot set, then click Add to add it to the project (Figure 5-1). Figure 5-1 Import from 4700 Proteomics Analyzer Dialog Box To delete a selected spot set from the sample set, select Remove. Note: A spot set cannot be deleted from a sample set after an analysis has been associated with it, or the spot set has been submitted with the sample set for processing. 11. Repeat step 10 to add or delete additional spot sets. 12. Click OK. The selected spot sets are imported into the sample set in the GPS Explorer software database. Information about the sample set is displayed in the view area. 13. Click Save in the toolbar. GPS Explorer Getting Started Guide 5-3 July 18, :22 pm, Wallace_GS_Ch5.fm

77 Chapter 5 Protein ID by Sample Set (MS/MS Spectra Only) Create an Analysis To create an analysis: 1. Select, then right-click the 9 protein mix sample set in the Project view. 2. Enter a unique name for the analysis, or accept the default name. 3. Click Save to save your changes. Note: You do not need to specify the analysis type or flag samples for processing when the association is By Sample. By default, the system selects MS/MS as the analysis type, and all data are processed. 4. Click the Settings bar to open the Analysis Settings dialog box. 5. In the Search Settings tab, select Open Search Settings, then select 9 Protein Mix from the list to apply default search settings. The search settings are described in Table 5-1. Table 5-1 Search Settings for 9 Protein Mix Parameter Setting Taxonomy All entries Report Top... Hits 100 Note: The top hits to report is the maximum number of protein identifications that are returned.the actual number returned can be less because the protein identifications may be below significance before the maximum number of protein identifications is obtained. Database Enzyme SwissProt Trypsin Max. Missed Cleavages 1 Fixed Modifications Variable Modifications Selected Oxidation (M) Carbamidomethyl (C) 5-4 GPS Explorer Getting Started Guide July 18, :22 pm, Wallace_GS_Ch5.fm

78 Create an Analysis Monoisotopic or Average Peptide Tol Parameter Monoisotopic Note: Because isotopic peaks are resolved, you select Monoisotopic; if they were unresolved, you would select Average. 75 ppm Setting Peptide Charges 1+ Significant Threshold Optional or 85% Note: Significant Threshold is a filter that takes a statistical percent of the Mascot Mowse score (protein score or ion score) greater than the probability that the observed match is a random event and reports, in the Results Browser, the value as a percent confidence value. When a threshold is set, the Results Browser displays only results equal to or above the significant threshold. For example, with the threshold set to 5%, only the top 95% of results that meet the criteria are displayed. Species Information Required MS/MS Tol Selected. This setting displays the species information for all identified proteins. Note: Retrieving species information increases database search time. 0.3 Da 6. In the MS/MS Peak Filtering tab, select Open Peak Filtering Settings, then select General MS-MS to apply peak filtering settings. Settings are described in Table 5-2. Table 5-2 Peak Filter Settings for MS/MS Mass Range Parameter Setting 60 to 20 Da below the precursor mass Minimum S/N 10 Intensity Filter Used Selected GPS Explorer Getting Started Guide 5-5 July 18, :22 pm, Wallace_GS_Ch5.fm

79 Chapter 5 Protein ID by Sample Set (MS/MS Spectra Only) Parameter Setting Max. Number of Peaks 65 Note: This is the maximum number of peaks submitted for database searching, after all filtering has occurred. Peak Density Used Peak Density Selected 50 per 200 Da Note: This is the number of peaks per 200 Da that is used to create the mass list for database searching. 7. Click Save Settings to apply the settings. 8. When queried to skip base MS peak filtering settings, click Yes. 5-6 GPS Explorer Getting Started Guide July 18, :22 pm, Wallace_GS_Ch5.fm

80 Prepare and Submit Samples for Processing Prepare and Submit Samples for Processing To prepare and submit samples for processing: 1. Click Import Job Runs in the Sample Set List window. The Import From 4700 Proteomics Analyzer window opens (Figure 5-2). Figure 5-2 Import Job Runs Dialog Box Note: You can import base MS analyses with the MS/MS runs. However, the base MS data is used only with combined MS-MS/MS runs. 2. In the left pane, click for the appropriate 4700 Proteomics Analyzer to view all the projects in its database. 3. Click to expand the lists of analyses for the selected sample set. GPS Explorer Getting Started Guide 5-7 July 18, :22 pm, Wallace_GS_Ch5.fm

81 Chapter 5 Protein ID by Sample Set (MS/MS Spectra Only) 4. Select run number 2 from the list of available runs, then click Add. 5. Click OK to import the run. Because the experiment is associated by sample set, you do not need to select samples to submit. All data are processed. 6. Click Save to save your analysis. 7. Click in the toolbar to submit the samples for processing. Note: The server must be running before you submit the analysis for processing. Otherwise, the analysis remains in the waiting state. See Manage Database Functions on page B-5. Monitor Job Processing To monitor the progress of your job: 1. Click in the GPS Explorer toolbar. The Project view lists projects with sample sets and analyses that are ready for processing. The Job list shows the job queue. (Jobs that have been processed or are not ready for processing are not listed). In addition, the right side of the screen displays information on the selected analysis. For association by sample, the Sample Set Job Status table displays information about the sample (Figure 5-3 on page 5-9). 5-8 GPS Explorer Getting Started Guide July 18, :22 pm, Wallace_GS_Ch5.fm

82 Monitor Job Processing Figure 5-3 Sample Set Job Status Table 2. Navigate to the analysis you created earlier in this chapter by clicking in the Project view next to the sample set you created in the previous chapter. The analysis you submitted is displayed in the sample set under the project. In the Job Status view (Figure 5-4), information for the job is listed in the queue. The list displays the order in which jobs are processed. Figure 5-4 Job Manager Job List Status Display 3. With the job status Active, look at the Plate view on the right side of the screen. Colors indicate processing progress. GPS Explorer Getting Started Guide 5-9 July 18, :22 pm, Wallace_GS_Ch5.fm

83 Chapter 5 Protein ID by Sample Set (MS/MS Spectra Only) Note: If the server is shut down while processing a job, the job status is suspended. Processing does not restart until the server is running again. If the job fails, it remains in the queue with a job status of Failed. 4. Right-click the GPS Explorer toolbar, then select Event Log to view system information related to the job (Figure 5-5). Figure 5-5 Event Log After the job has finished processing, it is removed from the queue GPS Explorer Getting Started Guide July 18, :22 pm, Wallace_GS_Ch5.fm

84 View Results View Results To view the results of the analysis: 1. Click in the GPS Explorer toolbar. 2. In the Project view, click to expand the project containing the sample set and analysis. 3. To view the results, navigate to the 9 protein mix... analysis you submitted for processing, then do one of the following: Click the link show the results. Right-click the Project view, then select Open. Double-click the analysis in the Project view. By default, the Results Browser opens with the Protein Summary table, Plate view, and Spectrum view arranged from top to bottom on screen. 4. In the Protein Summary table, select the highest-ranked protein, Serotransferrin precursor (Figure 5-6 on page 5-12). Plate view displays each well that contains serotransferrin in colors that indicate the confidence level of the data. (To review the color codes, right-click the Plates tab, then select Peak Colors Legend.) GPS Explorer Getting Started Guide 5-11 July 18, :22 pm, Wallace_GS_Ch5.fm

85 Chapter 5 Protein ID by Sample Set (MS/MS Spectra Only) Well 62 Figure 5-6 Plate View with Serotransferrin Wells Indicated 5. In Plate view, double-click well 62, which displays with the color for data of the highest confidence. The spectrum (the first in a sequence if there are multiple spectrums) for well 62 is displayed in the Spectrum tab, and the Peptide table for Serotransferrin expands beneath it in the Protein Summary view (Figure 5-7 on page 5-13) GPS Explorer Getting Started Guide July 18, :22 pm, Wallace_GS_Ch5.fm

86 View Results 6. Click the highest ranking peptide in the Peptide table to display the peptide s ion fragments. These ion fragment peaks are added to the base serotransferrin spectrum in the Spectrum tab (Figure 5-7). Figure 5-7 Spectrum Updated with Ion Fragment Peaks of the Highest Ranking Peptide Depending on how they are calculated, the ion fragment peaks are colored red (Mascot matches only) or blue (Mascot and Data Explorer matches). The default ion fragment algorithm is Mascot Score Only. You can change the Results Browser ion fragment settings to change results in the Spectrum view. GPS Explorer Getting Started Guide 5-13 July 18, :22 pm, Wallace_GS_Ch5.fm

87 Chapter 5 Protein ID by Sample Set (MS/MS Spectra Only) Note: Mascot Score Only matches exactly the spectrum display with Mascot results, except some ammonium ions not in the Mascot results may also display in the spectrum. 7. Right-click the Spectrum tab, then select the Settings... option to open the Ion Fragment Settings dialog box (Figure 5-8). Figure 5-8 Ion Fragment Settings Dialog Box 8. To select other ions for display, deselect the Mascot Score Only check box to deactivate it and activate the Ions options, then select the ions you want to display in the spectrum. You can also change the mass tolerance (for the spectrum display only) to broaden the mass range and label more ion fragment peaks in the spectrum. Changing the mass tolerance does not affect the score. 9. In the Spectrum tab, drag across the peaks between 60 and 175 Da. The display zooms in on the selected peaks (Figure 5-9 on page 5-15) GPS Explorer Getting Started Guide July 18, :22 pm, Wallace_GS_Ch5.fm

88 View Results Figure 5-9 Zoomed Spectrum 10. Right-click to zoom out of the spectrum. 11. Click the Peaks of Spectrum tab to display the Peak list. 12. To view the Peak Color Legend, right-click the Spectrum tab, then select Peak Color Legend. The legend box shows the color representations used in the Spectrum tab. 13. Right-click the Peak list, select Select All, then right-click and select Copy. 14. Open Windows Notepad, then select Edit > Paste. The Peak list is pasted into Notepad. 15. Save and close the Notepad file. You can edit the file later to fix column alignment or add or delete data before importing the contents into a spreadsheet. 16. Select and right-click a spot, then select Open DB Results to view the Mascot results returned from the database search. For more information on using the Results Browser, see the GPS Explorer Software Online Help. For information on creating reports from your data, see Chapter 6, Creating Reports. GPS Explorer Getting Started Guide 5-15 July 18, :22 pm, Wallace_GS_Ch5.fm

89 Chapter 5 Protein ID by Sample Set (MS/MS Spectra Only) 5-16 GPS Explorer Getting Started Guide July 18, :22 pm, Wallace_GS_Ch5.fm

90 Creating Reports 6 6 This chapter describes how to: Design a Report Report a Sample Associated by Well Including All Wells Including Well Range Report a Sample Associated by Set Export Reports GPS Explorer Getting Started Guide 6-1 July 18, :22 pm, Wallace_GS_Ch6.fm

91 Chapter 6 Creating Reports This chapter describes how to use the GPS Explorer software Report Generator to create reports for MS and MS/MS analysis results with association by well or sample set. You can create and view multiple reports at one time. Each report is displayed in a separate window under the main display area. Design a Report To design a report: 1. Select File > Layout Setup to open the Layout Setup dialog box (Figure 6-1). Figure 6-1 Layout Setup Header/Footer Tab 6-2 GPS Explorer Getting Started Guide July 18, :22 pm, Wallace_GS_Ch6.fm

92 Design a Report 2. In the Selected Template drop-down list, select the type of template that matches your analysis: Protein-Peptide Summary by Well For MS, MS/MS, or MS/MS combined sample sets associated by well Protein-Peptide Summary by Sample Set For MS/MS analysis only Spectrum View Generates a Spectrum table for MS/MS analysis 3. In the Selected Area drop-down list, select an area in which to place a layout element. 4. In the Selected Title drop-down list, select information to display. If the item you select is an image, browse to the location in Image Location, then select the image file. 5. Click Set Area to set your design. 6. Repeat steps 3 through 5 for each area in which you want to place a layout element. 7. Select the Body tab, then select the check boxes that correspond to the columns you want to display in the report (Figure 6-2 on page 6-4). GPS Explorer Getting Started Guide 6-3 July 18, :22 pm, Wallace_GS_Ch6.fm

93 Chapter 6 Creating Reports Figure 6-2 Layout Setup Body Tab The status area at the bottom of the selection box displays the number of pixels that remain to display report elements. 8. Click Save when you finish designing your report. 6-4 GPS Explorer Getting Started Guide July 18, :22 pm, Wallace_GS_Ch6.fm

94 Report a Sample Associated by Well Report a Sample Associated by Well Including All Wells You can generate results for a sample associated by well to include: All wells A range of wells To generate a report that includes all wells: 1. In Project view, select the analysis of interest, then select Reports > Protein-Peptide Summary by Well. The Report Set Up dialog box for a Protein-Peptide Sample Associated by Well opens, displaying the project, sample set, and selected analysis (Figure 6-3). Figure 6-3 Report Setup By Well Dialog Box 2. Select Include All Wells. GPS Explorer Getting Started Guide 6-5 July 18, :22 pm, Wallace_GS_Ch6.fm

95 Chapter 6 Creating Reports 3. In the Order By drop-down list, select Rank, then select one of the following: Include All Rank Include Only Top, then enter the number of top-ranked proteins to include. 4. Select one or more of the following: Show Company Logo only on the first page Show Analysis Information only on the first page Include Protein Group Include Peptide Information 5. Click OK when you have finished.your report is generated and opens in the Report view area of the main screen. Including Well Range To generate a report that includes a range of wells: 1. In the Project view, select the analysis of interest. 2. Select Reports > Protein-Peptide Summary by Well. The Report Set Up dialog box for a Protein-Peptide Summary by Well opens, displaying the project, sample set, and selected analysis. 3. Select Selected Wells. The Well list box is activated, allowing you to specify any range of contiguous and noncontiguous wells in your sample. 4. To add wells to the list, click to open the Well Selection dialog box (Figure 6-4). Figure 6-4 Well Selection Dialog Box 6-6 GPS Explorer Getting Started Guide July 18, :22 pm, Wallace_GS_Ch6.fm

96 Report a Sample Associated by Well 5. Select Single Well or Well Range, then select the plate and well information. For single wells, select the Plate and Well. For a range of wells, select the Beginning Plate and Well, then select the Ending Plate and Well. 6. Click OK. The well or range is displayed in the Well list box. 7. Repeat steps 3 through 5 to add more well groups. To delete an entry from the well list, select the entry in the list, then click. 8. In the Order By drop-down list, select Rank, then select one of the following: Include All Rank. Include Only Top, then enter the number of top-ranked proteins to include. 9. Select one or more of the following: Show Company Logo only on the first page Show Analysis Information only on the first page Include Protein Group Include Peptide Information 10. Click OK. Your report is generated and opens in the Report view area of the main screen. GPS Explorer Getting Started Guide 6-7 July 18, :22 pm, Wallace_GS_Ch6.fm

97 Chapter 6 Creating Reports Report a Sample Associated by Set To generate a report for a sample associated by set: 1. In the Project view, select the analysis of interest. 2. Select Reports > Protein Summary by Sample Set. The Report Set Up dialog for a Protein-Peptide Sample Associated by Set opens, displaying the project, sample set, and selected analysis. 3. In the Order By drop-down list, select Rank, then select one of the following: Include All Rank. Include Only Top, then enter the number of top-ranked proteins to include. 4. Select one or more of the following: Show Company Logo only on the first page Show Analysis Information only on the first page Include Protein Group Include Peptide Information 5. Click OK. Your report is generated and opens in the Report view area of the main screen. 6-8 GPS Explorer Getting Started Guide July 18, :22 pm, Wallace_GS_Ch6.fm

98 Export Reports Export Reports Note: You must generate a report before you can export it. To export a report to a format that you can use in other applications: 1. Select File > Export Data. The Export Data dialog box opens (Figure 6-5). Figure 6-5 Export Data Dialog Box 2. Browse to the location where you want the report data exported. 3. In the Save as type field, select the file format in which you want to export the report:.xls for Microsoft Excel.PDF for Acrobat Reader.RTF for Rich Text Format.TXT for plain text.html for Browser compatibility 4. Enter an appropriate filename. 5. Click Save. GPS Explorer Getting Started Guide 6-9 July 18, :22 pm, Wallace_GS_Ch6.fm

99 Chapter 6 Creating Reports 6-10 GPS Explorer Getting Started Guide July 18, :22 pm, Wallace_GS_Ch6.fm

100 Displaying Information A A This appendix describes how to: Set the Well Scale A-2 Display User-Defined Sample Information A-2 Customize Well-Color Display A-5 GPS Explorer Getting Started Guide A-1 July 18, :22 pm, Wallace_GS_AppA.fm

101 Appendix A Displaying Information Set the Well Scale To change the spot size of the well grid relative to the size as specified in the plate configuration information: 1. Click Sample Set Up in the GPS Explorer toolbar. 2. Right-click the well grid, select Well Scale Setting, then enter a percentage value (Figure A-1). Figure A-1 Plate Scale Factor Setting Dialog Box Display User-Defined Sample Information In the Configuration application, an administrator can define up to 10 text fields that can be assigned to each sample spot in the sample list. Text can be entered manually from the sample list. Alternatively, text can be imported from an ASCII text file. For information on specifying user-defined sample information columns, see Configure the Desktop on page B-2. Display User-Defined Columns To display user-defined columns: 1. In the Sample Setup application, right-click the Sample list, then select Column Visibility. The User Defined Sample Information Visibility dialog box displays the available user-defined columns (Figure A-2 on page A-3). A-2 GPS Explorer Getting Started Guide July 18, :22 pm, Wallace_GS_AppA.fm

102 Display User-Defined Sample Information Figure A-2 User Defined Sample Information Visibility Dialog Box Import Column Information 2. Select the boxes that correspond to the columns you want to display. 3. Click OK to display the new columns in the Sample list. To import column information from a text file: 1. In the Sample List toolbar, select. 2. Select import parameters as described in Table A-1. Table A-1 Import Parameters for User-Defined Columns Filename Parameter Skip Header Row Description Enter the name of or browse to the text file to be imported. Specifies that the import skip the first row, which will be treated as the header row in the text file. GPS Explorer Getting Started Guide A-3 July 18, :22 pm, Wallace_GS_AppA.fm

103 Appendix A Displaying Information Parameter Filename Available Columns Add Remove Selected Columns Move Up Move Dn File Format Description Enter the name of or browse to the text file to be imported. Lists all the columns that are available for import. Allows you to skip columns if the text file has additional information that is not to be imported. Moves the selected column on the left into the Selected Columns list on the right. Moves the selected columns out of the Selected Columns list. Lists the columns that the text file contains. Allows you to skip columns if the text file has additional information that is not to be imported. Moves the selected column name up in the list. Moves the selected column name down in the list. Allows you to select either Comma-Separated Values or Tab-Delimited Text. 3. Click OK to accept the settings and import the selected file. A-4 GPS Explorer Getting Started Guide July 18, :22 pm, Wallace_GS_AppA.fm

104 Customize Well-Color Display Customize Well-Color Display To set well color match criteria: 1. In the Results Browser, select View > Plate > Settings. The Plate Settings dialog box opens. 2. Select Protein Identification. 3. Click a color box to display the Color palette (Figure A-3). Figure A-3 Color Dialog Box 4. Select a color on the palette. 5. Click OK. GPS Explorer Getting Started Guide A-5 July 18, :22 pm, Wallace_GS_AppA.fm

105 Appendix A Displaying Information A-6 GPS Explorer Getting Started Guide July 18, :22 pm, Wallace_GS_AppA.fm

106 Managing the GPS Explorer System B B This appendix describes how to: Configure the Desktop B-2 Configure the Instrument B-4 Manage Database Functions B-5 Manage Users B-6 GPS Explorer Getting Started Guide B-1 July 18, :22 pm, Wallace_GS_AppB.fm

107 Appendix B Managing the GPS Explorer System Configure the Desktop You can customize the appearance of your computer desktop by: Selecting the style of the buttons on your desktop toolbar Specifying project view sorting options Enabling multiple Results Browsers Specifying the tiling layout of the desktop To configure your desktop: 1. Click in the GPS Explorer toolbar. 2. In the Desktop Configuration tab, select and deselect Flat Toolbar Buttons to switch the GPS Explorer toolbar buttons between 3-D and 2-D. 3. In the Toolbar Button Style drop-down list, select one of the following: Large Icon with Text Large Icon Small Icon 4. In the Toolbar Orientation drop-down list, set the orientation to Horizontal or Vertical. 5. Select Global Project View Sorting, then select one of the sorting options for the Project view: Node name User name Creation time Modification time Note: If you do not specify the sort order in this field, you can specify the sort order individually in each GPS Explorer application. 6. In the Desktop Tiling drop-down list, select one of the tiling options to display multiple applications open on the desktop: None Horizontal Vertical B-2 GPS Explorer Getting Started Guide July 18, :22 pm, Wallace_GS_AppB.fm

108 Configure the Desktop Cascade Maximize 7. Select Multiple Results Browsers, then specify the number of Results Browser windows you can open at one time (up to a maximum of 5) by clicking. 8. Click the User Defined Columns Setup button to open the User Defined Sample Information Fields dialog box (Figure B-1), where you configure the sample information displayed for each sample spot. Figure B-1 User-Defined Sample Information Fields Dialog Box 9. To define up to 10 sample information fields, click the User Field check box for each field you want to define, then enter a column header. Each defined field is associated with the sample list in each application. GPS Explorer Getting Started Guide B-3 July 18, :22 pm, Wallace_GS_AppB.fm

109 Appendix B Managing the GPS Explorer System Configure the Instrument A user with administrative privileges can add, delete, or disable connections between the GPS Explorer software and the 4700 Proteomics Analyzer. To add, edit, or disable 4700 Proteomics Analyzer connections: 1. Click in the GPS Explorer toolbar. 2. Select the Instrument Configuration tab. 3. To add or edit a connection, click Add or Edit. The Add or Edit Instrument dialog box opens (Figure B-2). Figure B-2 Edit Instrument Dialog Box 4. To add an instrument to your configuration, type the computer name of the 4700 Proteomics Analyzer in the Instrument name field. (Spaces are not allowed.) To edit an existing instrument connection, select the instrument, then do one of the following: To change an instrument connection, type the change in the Instrument name field, To disable an instrument connection, select Disabled. 5. Click OK. The GPS Explorer software determines the validity of the connection. If the instrument is not found, a message box prompts you to add the instrument. When the instrument is added to the list, you can browse the instrument for spot sets and runs to import. B-4 GPS Explorer Getting Started Guide July 18, :22 pm, Wallace_GS_AppB.fm

110 Manage Database Functions 6. In the Instrument Configuration tab, click Update to apply and save the changes, or click the Instrument Configuration tab to exit Instrument Configuration without applying the changes. Manage Database Functions Users with administrative privileges can perform database administrative functions in the GPS Explorer software. To manage GPS Explorer database functions: 1. Click in the GPS Explorer toolbar. 2. Select the System Maintenance tab (Figure B-3). 3. To activate Database Management functions, select Set Server Off. If a job is currently processing, you can select to cancel, shut down immediately, or shut down after the job has finished processing. Figure B-3 GPS Explorer Configuration Dialog Box GPS Explorer Getting Started Guide B-5 July 18, :22 pm, Wallace_GS_AppB.fm

111 Appendix B Managing the GPS Explorer System 4. In the Database Management section, select: Backup To back up the contents of the database into a data file. Restore To restore the content of the database from a backup data file. Purge To purge or clean up the database content. Extend To extend the database space to additional hard drives. Manage Users To manage GPS Explorer system users: 1. Click in the GPS Explorer toolbar. 2. Select the User Administration tab to display a list box showing the available users in the system (Figure B-4). Figure B-4 User Administration Tab B-6 GPS Explorer Getting Started Guide July 18, :22 pm, Wallace_GS_AppB.fm

112 Manage Users 3. To edit a user, select the user, then click Edit, and change the user information. 4. To add a user, click Add, then type any of the following: User Name Enter a unique name (3 characters minimum) for the user. First Name Enter the user s first name (3 characters minimum). Last Name Enter the user s last name (3 characters minimum). 5. Select an Account Type: Administrator Access to all operations and data through the system, including user and database administration activities. Privileged Access to all data but cannot perform user and database administrative operations. Standard Access to data created by the user only. 6. Enter and confirm a user password. Password If a new user is being added, type the password in this field. An * appears in the field for each letter typed (4 characters minimum, 16 characters maximum). Confirm Password Retype the new password in this field. The field remains inactive until the correct password and name are entered in the appropriate fields, and the user has entered something in the New Password edit box. An * appears in the field for each character typed. 7. Select Account Disabled to enable or disable a selected account. Click the box to disable. You cannot disable a user who is currently logged in. Note: You cannot remove a user because the username is associated with data stored in the database. 8. Click OK to accept the changes and return to the User dialog box, or click Cancel to cancel the changes and return to the User dialog box. GPS Explorer Getting Started Guide B-7 July 18, :22 pm, Wallace_GS_AppB.fm

113 Appendix B Managing the GPS Explorer System Viewing the User Audit Trail 1. Click in the GPS Explorer toolbar. 2. Select the User Administration tab, then click the Audit Trail button to display the User Audit Trail dialog box (Figure B-5). Figure B-5 User Audit Trail Dialog Box In the User Audit Trail dialog box, a user with administrative privileges can: Add users Change passwords Change user names Enable and disable user accounts B-8 GPS Explorer Getting Started Guide July 18, :22 pm, Wallace_GS_AppB.fm