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© 2009 Thermo Fisher Scientific Inc. All rights reserved.
Xcalibur is a registered trademark of Thermo Fisher Scientific Inc. in the United States. ZCore and Proteome
Discoverer are trademarks of Thermo Fisher Scientific Inc. in the United States.
Microsoft, Excel, Windows, and Windows Vista are registered trademarks of Microsoft Corporation in the
United States and other countries.
All other trademarks are the property of Thermo Fisher Scientific Inc. and its subsidiaries.
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 contract 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: Revision A October 2009
Software Version: Proteome Discoverer 1.1
For Research Use Only. Not for use in diagnostic procedures.
Thermo Scientific Proteome Discoverer User Guide iii
C
Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xi
Related Documentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .xi
System Requirements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xii
Obtaining a License . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xii
Special Notices . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .xiv
Contacting Us . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .xiv
Chapter 1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1
Features. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Search Engines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
Wizards and Workflow Editor. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
Quantitation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Qual Browser . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
Peptides and Fragment Ions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
Fragmentation Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
MudPIT Experiments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
Workflow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
Inputs and Outputs. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
FASTA Databases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
Inputs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
Outputs. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
Limitations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
Chapter 2 Using the Proteome Discoverer Interface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .13
Proteome Discoverer User Interface. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
Main Window. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
Colors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
Menu Bar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
Shortcut Menus. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
Toolbar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
Job Queue . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
Using Search Results Window Icons . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26
Using Dialog Box Icons. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28
Contents
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iv Proteome Discoverer User Guide Thermo Scientific
Customizing the User Interface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28
Customizing the Toolbar. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28
Customizing Menus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36
Customizing the Job Queue Toolbar. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44
Chapter 3 Getting Started. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .47
Starting a New Search by Using the Search Wizards . . . . . . . . . . . . . . . . . . . . . 47
Configuring Search Parameters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48
Starting a New Search . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53
Search Wizard Dialog Box Parameters. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66
Starting a New Search by Using the Workflow Editor. . . . . . . . . . . . . . . . . . . . 71
Workflow Editor Parameters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73
Creating a Search Workflow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73
Opening an Existing Workflow. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82
Deleting an Existing Workflow Template . . . . . . . . . . . . . . . . . . . . . . . . . . . 85
Changing the Name and Description of a Workflow Template. . . . . . . . . . . 85
Importing a Workflow from an XML Template . . . . . . . . . . . . . . . . . . . . . . 86
Importing Workflows in Other Formats . . . . . . . . . . . . . . . . . . . . . . . . . . . . 86
Workflow Nodes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87
Adding a Non-Fragment Filter Node for High-Resolution Data . . . . . . . . . . 90
Saving a Workflow as an XML Template . . . . . . . . . . . . . . . . . . . . . . . . . . . 92
Exporting Spectra . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 92
Creating a Search Workflow for Multiple .raw Files from the Same
Sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 94
Creating a Quantitation Workflow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 96
Creating Parallel Workflows . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 96
Using the Discoverer Daemon Utility . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 98
Starting Discoverer Daemon in a Window . . . . . . . . . . . . . . . . . . . . . . . . . . 98
Selecting the Server . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 98
Starting a Workflow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 99
Creating a Parameter File for Use with the Xcalibur Data System . . . . . . . . 102
Monitoring Job Execution in Discoverer Daemon. . . . . . . . . . . . . . . . . . . . 103
Discoverer Daemon Window Parameters . . . . . . . . . . . . . . . . . . . . . . . . . . 105
Logging on to a Remote Server . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 107
Running Discoverer Daemon from the Xcalibur Data System. . . . . . . . . . . 107
Running Discoverer Daemon on the Command Line . . . . . . . . . . . . . . . . . 128
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Thermo Scientific Proteome Discoverer User Guide v
Chapter 4 Searching for Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .131
Using FASTA Databases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 131
Displaying FASTA Files . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 131
Adding FASTA Files . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 133
Deleting FASTA Files . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 133
Adding a Protein Sequence and Reference to a FASTA Database File . . . . . 133
Finding Protein Sequences and References . . . . . . . . . . . . . . . . . . . . . . . . . 135
Compiling a FASTA Database. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 140
Excluding Individual Protein References and Sequences from a FASTA
Database. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 144
FASTA Database Utilities Dialog Box Parameters . . . . . . . . . . . . . . . . . . . . 145
Enter Filter Criteria for Reference Dialog Box Parameters. . . . . . . . . . . . . . 149
Managing FASTA Indexes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 149
Modifying Chemicals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 163
Dynamic Modifications. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 163
Static Modifications. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 164
Opening the Chemical Modifications View. . . . . . . . . . . . . . . . . . . . . . . . . 164
Adding Chemical Modifications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 166
Adding Amino Acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 167
Deleting Chemical Modifications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 168
Importing Chemical Modifications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 169
Deleting Amino Acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 170
Using Qual Browser . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 170
Customizing Cleavage Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 172
Cleavage Reagents View Parameters. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 173
Adding a Cleavage Reagent . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 176
Deleting a Cleavage Reagent . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 176
Modifying a Cleavage Reagent. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 176
Filtering Cleavage Reagent Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 176
Chapter 5 Interpreting Search Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .179
Using the Results Report . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 179
Opening .msf Files . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 181
Saving .msf Files . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 187
Closing .msf Files . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 188
Importing .srf Files . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 188
Organizing Reports . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 188
Controlling the Search Report Windows. . . . . . . . . . . . . . . . . . . . . . . . . . . 201
Using the Search Report Menu . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 201
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vi Proteome Discoverer User Guide Thermo Scientific
Using the Proteins Page. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 201
Displaying the Proteins Page . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 202
Researching Groups of Proteins. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 206
Interpreting Your Results with the Chromatogram View. . . . . . . . . . . . . . . 207
Interpreting Your Results with the Protein Identification Details View . . . . 210
Interpreting Your Results with the Report Item Distribution Chart . . . . . . 215
Interpreting Your Results with the Sequence Comparison View . . . . . . . . . 222
Using the Peptides Page. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 224
Displaying the Peptides Page . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 225
Interpreting Your Results with the Chromatogram View. . . . . . . . . . . . . . . 231
Interpreting Your Results with the Extracted Ion Chromatogram View. . . . 234
Interpreting Your Results with the Peptide Identification Details View . . . . 237
Interpreting Your Results with the Peptide Consensus View . . . . . . . . . . . . 246
Interpreting the Dual-Peptide Consensus View . . . . . . . . . . . . . . . . . . . . . . 254
Displaying Post-Translational Modification Information in the
UNIMOD Web View . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 257
Interpreting Your Results with the Isotope Pattern View . . . . . . . . . . . . . . . 260
Interpreting Your Results with the Spectrum View . . . . . . . . . . . . . . . . . . . 262
Interpreting Your Results with the Fragment Match Spectrum View. . . . . . 264
Using the Search Input Page . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 265
Search Input Page Parameters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 266
Using the Result Filters Page . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 268
Using the Peptide Confidence Page. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 268
Using the Search Summary Page . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 268
Using the Quantitation Summary Page . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 270
Creating a Report from Multiple Results. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 270
Finding Common or Unique Proteins in Multiple Searches . . . . . . . . . . . . 272
Applying Filters Specific to Different Searches in Multiconsensus
Reports. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 275
Chapter 6 Filtering Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .277
Result Filters Page . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 277
Displaying the Result Filters Page . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 277
Result Filters Page Parameters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 278
Filtering Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 282
Filtering Results with the Filters on the Result Filters Page . . . . . . . . . . . . . 282
Peptide Filters in SEQUEST Reports . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 290
Peptide Filters in Mascot Reports . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 295
Peptide Filters in ZCore Reports . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 299
Filtering Results with Row Filters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 302
Displaying Filtered-Out Rows. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 310
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Thermo Scientific Proteome Discoverer User Guide vii
Calculating False Discovery Rates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 311
Target False Discovery Rates (FDRs). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 312
Peptide Confidence Indicators. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 312
Setting Up False Discovery Rates in Search Wizards and the
Workflow Editor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 313
Viewing the Results on the Peptide Confidence Page . . . . . . . . . . . . . . . . . 315
Recalculating the False Discovery Rates. . . . . . . . . . . . . . . . . . . . . . . . . . . . 317
Peptide Confidence Page Parameters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 319
Chapter 7 Quantitation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .323
Activating the Quantitation Menu . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 323
Performing Isobarically Labeled Quantitation . . . . . . . . . . . . . . . . . . . . . . . . . 324
TMT Quantitation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 324
iTRAQ Quantitation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 325
Performing TMT and iTRAQ Quantitation . . . . . . . . . . . . . . . . . . . . . . . . 325
Performing TMT Quantitation on HCD and CID Scans . . . . . . . . . . . . . . 329
Setting Up the Quantitation Method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 330
Specifying the Quantitation Channels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 330
Setting Up Quantitation Channels for Ratio Reporting. . . . . . . . . . . . . . . . 333
Setting Up the Ratio Calculation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 335
Setting Peptide Parameters Used to Calculate Protein Ratios. . . . . . . . . . . . 337
Correcting Experimental Bias . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 338
Checking the Quantitation Method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 340
Restoring Quantitation Template Defaults . . . . . . . . . . . . . . . . . . . . . . . . . 340
Setting Up the Quantitation Method for Multiple Input Files . . . . . . . . . . 340
Processing Method Editor Dialog Box Parameters. . . . . . . . . . . . . . . . . . . . 344
Excluding Peptides from the Protein Quantitation . . . . . . . . . . . . . . . . . . . 349
Correcting for Impurities by Using Reporter Ion Isotopic Distribution
Values . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 349
Adding a Quantitation Method. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 351
Quantitation Methods View Parameters . . . . . . . . . . . . . . . . . . . . . . . . . . . 354
Create Quantitation Method Dialog Box Parameters . . . . . . . . . . . . . . . . . 354
Changing a Quantitation Method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 355
Removing a Quantitation Method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 356
Summarizing the Quantitation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 356
Calculating Peptide Ratios. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 358
Understanding the Peptide Ratio Distribution Chart . . . . . . . . . . . . . . . . . 359
Handling Missing and Extreme Values in Calculating Peptide Ratios . . . . . 361
Displaying Quantitation Channel Values . . . . . . . . . . . . . . . . . . . . . . . . . . . . 363
Displaying Quantitation Channel Values for Isobarically Labeled
Quantitation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 364
Quantitation Channel Values Chart Parameters . . . . . . . . . . . . . . . . . . . . . 366
Contents
viii Proteome Discoverer User Guide Thermo Scientific
Displaying the Quantitation Spectrum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 366
Displaying the Quantitation Spectrum Chart for Isobarically Labeled
Quantitation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 366
Quantitation Spectrum Chart Parameters . . . . . . . . . . . . . . . . . . . . . . . . . . 372
Calculating Ratio Count and Variability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 372
Replicates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 373
Treatments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 373
Ratio Count. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 373
Ratio Variability. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 373
Calculating and Displaying Protein Ratios for Multiconsensus Reports. . . . . . 375
Calculating Protein Ratios in Multiconsensus Reports Treated as
Treatments. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 377
Calculating Protein Ratios in Multiconsensus Reports Treated as
Replicates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 377
Mixed Mode . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 379
Troubleshooting Quantitation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 379
Chapter 8 Exporting Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .383
Export Options . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 383
Exporting Spectra . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 384
Export Spectra Dialog Box Parameters . . . . . . . . . . . . . . . . . . . . . . . . . . . . 385
Exporting Spectra Without a Peptide Match . . . . . . . . . . . . . . . . . . . . . . . . . . 386
Exporting Search Results to an .xml File . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 387
Export to ProtXML Dialog Box Parameters . . . . . . . . . . . . . . . . . . . . . . . . 388
Copying or Saving a View to an Image . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 388
Exporting Exclusion Mass Lists to Xcalibur. . . . . . . . . . . . . . . . . . . . . . . . . . . 389
Parameters on the Export Exclusion/Inclusion List Dialog Box . . . . . . . . . . 392
Exporting Search Results to Excel . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 393
Export to Excel Workbook Dialog Box Parameters . . . . . . . . . . . . . . . . . . . 396
Exporting Protein References to a FASTA File . . . . . . . . . . . . . . . . . . . . . . . . 397
Export to FASTA Dialog Box Parameters . . . . . . . . . . . . . . . . . . . . . . . . . . 398
Chapter 9 Reference . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .399
File Menu . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 400
Search Report Menu . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 401
Quantitation Menu . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 405
Processing Menu . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 406
Workflow Editor Menu. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 406
Administration Menu . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 408
Tools Menu. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 410
Window Menu . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 410
Help Menu . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 411
Contents
Thermo Scientific Proteome Discoverer User Guide ix
Shortcut Menus. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 411
Proteins Page Shortcut Menu . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 411
Peptides Page Shortcut Menu . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 413
Search Input Page Shortcut Menu. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 416
Graph Shortcut Menu . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 417
Workflow Editor Nodes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 418
Data Input Nodes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 418
Spectrum And Feature Retrieval Nodes . . . . . . . . . . . . . . . . . . . . . . . . . . . . 419
Spectrum Processing Nodes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 425
Spectrum Filters Nodes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 436
Peptide/Protein ID Nodes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 440
Quantitation Nodes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 453
Data Export Node. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 456
Appendix A FASTA Reference . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .459
FASTA Databases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 459
NCBI . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 459
MSIPI . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 460
IPI. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 460
UniRef100 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 460
SwissProt and TrEMBL. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 461
MSDB. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 461
Custom Database Support. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 462
Custom Parsing Rule A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 462
Custom Parsing Rule B . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 462
Custom Parsing Rule C . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 463
Appendix B Chemistry References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .465
Amino Acid Mass Values. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 465
Enzyme Cleavage Properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 466
Fragment Ions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 467
Appendix C Migrating from the BioWorks to the Proteome Discoverer Application . . . . .469
Configuring SRF Importer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 469
Installing the Software for the Migration. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 469
Preparing the Computer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 470
Installing BioWorks Software on a Reserved PC . . . . . . . . . . . . . . . . . . . . . 470
Installing the Proteome Discoverer Application. . . . . . . . . . . . . . . . . . . . . . 471
Adding FASTA Databases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 471
Installing a Workflow Template . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 471
Contents
xProteome Discoverer User Guide Thermo Scientific
Converting .srf, .dta, .out, and .raw Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . 473
Moving BioWorks 3.3.1 .srf Files to the Proteome Discoverer
Application. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 474
Moving BioWorks 3.3 or Earlier .srf Files to the Proteome Discoverer
Application. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 480
Importing BioWorks .dta, .out, and FASTA files into the Proteome
Discoverer Application. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 484
Importing .raw Data Files into the Proteome Discoverer Application . . . . . 489
Possible Causes of Failed Migrations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 491
Known Issues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 494
Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .497
Thermo Scientific Proteome Discoverer User Guide xi
P
Preface
This guide describes how to use the Proteome Discoverer™ 1.1 application for peptide and
protein mass spectrometry analyses.
To provide us with comments about this document, click the link below. Thank you in
advance for your help.
Related Documentation
The Proteome Discoverer application includes Help and these manuals as PDF files:
• Proteome Discoverer User Guide
• Proteome Discoverer Installation Guide
YTo view product manuals
•Proteome Discoverer User Guide: Go to Start > Programs > Thermo Proteome
Discoverer > Proteome Discoverer 1.1 User Guide.
•Proteome Discoverer Installation Guide: Go to Start > Programs > Thermo Proteome
Discoverer > Proteome Discoverer 1.1 Installation Guide.
Contents
•Related Documentation
•System Requirements
•Obtaining a License
•Special Notices
•Contacting Us
Preface
System Requirements
xii Proteome Discoverer User Guide Thermo Scientific
YTo open Help
• From the main Proteome Discoverer window, choose Help > Help Contents.
• If available for a specific window or view, click Help or press F1 for information
about setting parameters.
For more information, visit www.thermo.com. You can find application notes at
www.thermo.com/appnotes.
System Requirements
The Proteome Discoverer application requires a license. In addition, your system must meet
these minimum requirements.
Obtaining a License
Follow this procedure to obtain licenses for the Proteome Discoverer application.
YTo request your Proteome Discoverer license
1. From the Proteome Discoverer main menu bar, choose Administration > Manage
Licenses.
2. Verify that the bar code is listed in the Barcode column in the row of the feature that you
want the license for, as shown in the example in Figure 1. If it is not, enter the number
from the Proteome Discoverer application jewel case.
The bar code is read-only for permanent licenses.
System Requirements
Hardware • 2 GHz processor with 2 GB RAM
•DVD-ROM drive
• Video card and monitor capable of 1280 × 1024 resolution (XGA)
• Screen resolution of 96 dpi
• 75 GB available on the C: drive
•NTFS format
Software • Microsoft™ Windows™ XP 32/64 Professional (English version)
with current service pack
• Microsoft Windows Vista 32/64 Business (English version) with
current service pack
Note Ensure that port 28199 is not blocked by firewalls.
Preface
Obtaining a License
Thermo Scientific Proteome Discoverer User Guide xiii
Figure 1. License Administration list of features
3. In the User Information area, type your contact information, as shown in Figure 2.
Figure 2. User Information area
4. In the Host Information area, shown in Figure 3, click Compose Email.
Figure 3. Host Information area
Your default e-mail tool creates a new e-mail message addressed to Thermo Fisher
Scientific requesting a Thermo Proteome Discoverer application license. If it cannot
automatically create the e-mail, click Copy to Clipboard and paste the
request-for-license text into a new e-mail.
5. If needed, request other licenses in this e-mail.
6. Send the e-mail to licenses.ms@thermo.com.
7. When the license is sent back to you, copy and paste all the sent text into the Add
Licenses box, as shown in Figure 4.
Preface
Special Notices
xiv Proteome Discoverer User Guide Thermo Scientific
Figure 4. Add Licenses window
8. Click Add Licenses.
9. Close and restart the Proteome Discoverer application.
Special Notices
Make sure you follow the precautionary statements presented in this guide. Special notices
appear in boxes.
Contacting Us
There are several ways to contact Thermo Fisher Scientific for the information you need.
YTo contact Technical Support
Find software updates and utilities to download at mssupport.thermo.com.
IMPORTANT Highlights information necessary to prevent damage to software, loss of
data, or invalid test results; or might contain information that is critical for optimal
performance of the system.
Note Highlights information of general interest.
Tip Highlights helpful information that can make a task easier.
Phone 800-532-4752
Fax 561-688-8736
E-mail us.techsupport.analyze@thermofisher.com
Knowledge base www.thermokb.com
Preface
Contacting Us
Thermo Scientific Proteome Discoverer User Guide xv
YTo contact Customer Service for ordering information
YTo copy manuals from the Internet
Go to mssupport.thermo.com and click Customer Manuals in the left margin of the
window.
YTo suggest changes to documentation or to Help
• Fill out a reader survey online at
http://www.surveymonkey.com/s.aspx?sm=R7gKOvhLXn3NTkpK2BefHQ_3d_3d.
• Send an e-mail message to the Technical Publications Editor at
techpubs-lcms@thermofisher.com.
Phone 800-532-4752
Fax 561-688-8731
E-mail us.customer-support.analyze@thermofisher.com
Web site www.thermo.com/ms
Thermo Scientific Proteome Discoverer User Guide 1
1
Introduction
This chapter introduces you to the Proteome Discoverer application and describes its features
and functionality.
Features
The Proteome Discoverer application identifies proteins from the mass spectra of digested
fragments. It compares the raw data from mass spectrometry to the information from the
selected FASTA database. You can use these applications to analyze spectral data from all
Thermo Scientific and other mass spectrometers. Specifically, the Proteome Discoverer
application does the following:
• Works with peak-finding search engines such as SEQUEST™, Mascot™, and ZCore™ to
process all data types collected from low- and high-mass-accuracy mass spectrometry
(MS) instruments. The peak-finding algorithm searches the raw mass spectrometry data
and generates a peak list and relative abundances. The peaks represent the fragments of
peptides for a given mass and charge.
• Produces complementary data from a variety of dissociation methods and data-dependent
stages of tandem mass spectrometry.
• Combines, filters, and annotates results from several database search engines and from
multiple analysis iterations. The search engines correlate the uninterrupted tandem mass
spectra of peptides with databases, such as FASTA. See “Using FASTA Databases” on
page 131.
The Proteome Discoverer application includes the following features:
• Multiple search engines. As just noted, the Proteome Discoverer application includes the
SEQUEST, Mascot, and ZCore search engines. For information on these tools, see
“Starting a New Search by Using the Search Wizards” on page 47.
Contents
•Features
•Workflow
•Inputs and Outputs
•Limitations
1 Introduction
Features
2Proteome Discoverer User Guide Thermo Scientific
• The Workflow Editor for searching with multiple algorithms and merging results from
multiple dissociation techniques. See “Starting a New Search by Using the Workflow
Editor” on page 71.
• Support for isobarically labeled (iTRAQ and TMT) quantitation. See “Performing
Isobarically Labeled Quantitation” on page 324.
• Proteome Discoverer Daemon, which can perform multiple searches on multiple .raw
files at any given time. You can use it to perform searches on multiple .raw files taken
from multiple samples or replicates from the same sample. See “Using the Discoverer
Daemon Utility” on page 98.
• A number of graphical views that contain detailed information about the selected
peptides and proteins. You can display more than one view to perform a comparative
analysis of your selected peptide or proteins. See “Interpreting Search Results” on
page 179.
• The presentation of database search results available from multiple .raw files in a single
protein or peptide report. See “Creating a Search Workflow” on page 73.
• Support for FASTA databases and indexes. See “Using FASTA Databases” on page 131.
• The ability to import protein and peptide reports in standard spectrum data formats, such
as .mzData, .mzXML, and .mgf. See “Importing Workflows in Other Formats” on
page 86.
• The ability to export protein and peptide reports in standard spectrum data formats, such
as .mzData, .dta, and .mgf. “Exporting Data” on page 383 describes how to export your
data to these formats.
• The ability to export all or filtered results to protXML format. See “Exporting Search
Results to an .xml File” on page 387.
• The ability to merge filtered or unfiltered search results. See “Creating a Report from
Multiple Results” on page 270.
Search Engines
The Proteome Discoverer application includes the SEQUEST, Mascot, and ZCore search
engines; each produces complementary data. ZCore and SEQUEST are peptide search
engines distributed by Thermo Scientific. Mascot is a protein identification search engine
created by Matrix Science.
Mascot uses mass spectrometry data to identify proteins from primary sequence databases.
ZCore is specifically developed and optimized to evaluate both high-mass-accuracy and
low-mass-accuracy ETD data. SEQUEST can analyze three types.
• Electron-transfer dissociation (ETD)
• Electron-capture dissociation (ECD)
1 Introduction
Features
Thermo Scientific Proteome Discoverer User Guide 3
• Collision-induced dissociation (CID)
ETD and CID ion fragmentation methods produce distinct fragment ion sets:
• ETD and ECD generate primarily c and z fragment ions with preferences for precursor
ion charge states of +3 or higher.
• CID generates primarily b and y fragment ions with preferences for precursor ion charges
states of +3 or lower.
Frequently, peptides identified by CID are not observed with ETD and vice versa so that
combining results from CID and ETD can enhance sequence coverage. Many times CID and
ETD identify the same peptides, often with different precursor ion charge states. Combining
ETD and CID results improves confidence in identifications.
SEQUEST Search Engine
SEQUEST is specifically developed and optimized to evaluate both high-mass-accuracy and
low-mass-accuracy ETD, ECD, and CID data. You can use SEQUEST in combination with
automated LC-MS/MS and intelligent data acquisition tools to ensure the routine
identification of low-abundance proteins in complex mixtures.
The Proteome Discoverer application extracts relevant MS/MS spectra from the .raw file and
determines the precursor charge state and the quality of the fragmentation spectrum.
The SEQUEST search algorithm correlates experimental MS/MS spectra through
comparisons to theoretical in-silico peptide candidates derived from protein databases. The
proprietary cross-correlation identification algorithm at the core of SEQUEST uses a
sophisticated scoring system to help assess results. SEQUEST looks for characteristic spectral
patterns and then critically evaluates the equivalence of experimental and theoretical MS/MS
spectra. The identification algorithm extracts information and correctly identifies proteins
even when protein samples sizes are limited and the signal-to-noise ratio of spectra is low.
You can extract specific information from your results through the interactive data summary
screens. With a click, you can examine a fully annotated MS/MS spectrum, or view the
percent peptide coverage of an identified protein.
SEQUEST provides excellent search results on data acquired with Thermo Scientific ion trap
mass spectrometers. Using accurate mass windows decreases the search time, increases the
accuracy of the result, and decreases the false positive rate.
The Proteome Discoverer probability-based scoring system rates the relevance of the best
matches found by the SEQUEST algorithm. With this probability-based scoring, the
application can independently rank the peptides and proteins and increase the confidence in
protein identification. Additionally, this scoring system minimizes the time needed for data
interrogation or results review, increasing the overall throughput of the analysis.
1 Introduction
Features
4Proteome Discoverer User Guide Thermo Scientific
You can also automatically determine false discovery rates by comparing the results of forward
and reversed databases, which provides an additional means of increasing confidence in
protein identification.
Mascot Search Engine
Mascot uses mass spectrometry data to identify proteins from primary sequence databases.
For more details on Mascot, visit http://www.matrixscience.com.
ZCore Search Engine
ZCore is specifically developed and optimized to evaluate both high-mass-accuracy and
nominal-mass-accuracy ETD data. You can use ZCore to identify ETD MS/MS
fragmentation spectra of precursor ion charge states between 2 and 7.
The ZCore search algorithm correlates experimental MS/MS spectra through comparisons to
theoretical in-silico peptide candidates derived from protein databases, even when sample sizes
are limited and the signal-to-noise ratio of the spectra is low. ZCore evaluates the quality of
the fragmentation spectrum and scores the spectra.
You can extract specific information from your results through the interactive data summary
screens. With a click, you can examine a fully annotated MS/MS spectrum or view the
percent peptide coverage of an identified protein.
ZCore provides excellent search results on data acquired with Thermo Scientific ion trap mass
spectrometers.
To distinguish a correct peptide match from an incorrect peptide match, the ZCore algorithm
uses two calculations, a probability-based scoring of the fragment ion distribution and the
total fraction of covered fragment ion intensity. The score is reported as a probability (using a
-log10 value system), as well as an expectation value.
Wizards and Workflow Editor
You can use the Proteome Discoverer application’s search wizards or its Workflow Editor to
conduct data analysis searches of your spectra.
The search wizards are predefined to enable you to quickly set your search parameters and
obtain results. The Proteome Discoverer application includes a wizard for each of the supplied
search engines: ZCore, SEQUEST, and Mascot.
For information on how to use the wizards, see “Starting a New Search by Using the Search
Wizards” on page 47.
/
