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  5. BioWorks 3.3.1 SP1

Thermo Fisher Scientific BioWorks 3.3.1 SP1 User guide

  • Hello! I'm your chat assistant, and I've reviewed the BioWorks software XCALI 97194 User Guide. This document provides detailed instructions on using the software for protein identification and quantification, including features such as the SEQUEST search algorithm, PepQuan for quantification, and tools for data management. I'm ready to answer any questions you may have about the software and its functions.
  • What is the purpose of the BioWorks software?
    What is the SEQUEST search algorithm used for?
    What is PepQuan used for?
    What are Pepmap and Pepmatch used for?
For Research Use Only
Not for use in Diagnostic Procedures
XCALI-97194 Revision B May 2007
Xcalibur
BioWorks
User Guide
© 2007 Thermo Fisher Scientific Inc. All rights reserved.
Adobe® and Acrobat® are registered trademarks of Adobe Systems Incorporated. Microsoft®, Excel®, Windows®,
and ActiveX® are registered trademarks of Microsoft Corporation. SEQUEST®/SEQUEST® and ICAT® are
registered trademarks of the University of Washington. iTRAQ™ is a trademark of Applied Biosystems.
BLAST® is a registered trademark of the National Library of Medicine
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 released January 2007
Revision B released May 2007
For Research Use Only. Not regulated for medical or veterinary diagnostic use by U.S. Federal Drug
Administration or other competentauthorities.
Thermo Scientific manual-name iii
C
Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vii
About This Guide. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vii
Related Documentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vii
Safety and Special Notices . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vii
Contacting Us . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .viii
Assistance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .viii
Changes to the Manual and Online Help . . . . . . . . . . . . . . . . . . . . . . . . . . .viii
Chapter 1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1
Understanding BioWorks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
SEQUEST Search Algorithm . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
PepQuan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Results Interpretation Exporting and Reporting Data . . . . . . . . . . . . . . . . . . . 3
Pepmap . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Pepmatch . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
Additional Databases and Sequence Tools . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
What is New in BioWorks. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
Activation Type. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
Data Preprocessing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Using BioWorks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
BioWorks Work Flow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
BioWorks Input and Output File Types . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
Chapter 2 Setting your Default Configuration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .9
About Configuration. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Setting Default Parameters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
General Configuration Options. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
Default Folder . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
Internet URLs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
Mass Options . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
Trace Options . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
Enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
Modifications Menu . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
Static Modifications. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
Contents
Contents
iv manual-name Thermo Scientific
Search Engine Configuration Options. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
DTA Generation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
DTA Filters. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
FASTA Database Indexer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25
SEQUEST Search . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26
SEQUEST Cluster . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28
SEQUEST Sorcerer. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
Mascot Search . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30
Results Configuration Options . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
Results Table . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
Search Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32
Results Filters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34
Display Ions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36
PepQuan Configuration Options . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37
Isotope Labeling Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37
ICAT/c-ICAT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38
PepQuan iTRAQ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40
PepQuan SILAC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41
Peak Area/Height . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42
ICIS Peak Detection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43
Chapter 3 Performing Search . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .45
About SEQUEST . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45
Opening a RAW Data File . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46
Performing SEQUEST Search. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48
Setting Basic SEQUEST Search Options. . . . . . . . . . . . . . . . . . . . . . . . . . . . 48
Modifying the DTA Generation Parameters . . . . . . . . . . . . . . . . . . . . . . . . . 54
Setting the SEQUEST Search Parameters . . . . . . . . . . . . . . . . . . . . . . . . . . . 56
Using SEQUEST Batch Search . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62
Performing a SEQUEST Batch Search . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62
Searching MALDI Data With SEQUEST. . . . . . . . . . . . . . . . . . . . . . . . . . . 66
Creating a Sequence (.sld) File. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66
Searching vMALDI Samples in BioWorks. . . . . . . . . . . . . . . . . . . . . . . . . . . 68
Viewing MALDI Search Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71
Using Mascot Search . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72
Performing a Mascot Search . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72
Chapter 4 Viewing and Interpreting Search Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .77
Example Files . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77
About SEQUEST Search Results. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 78
Scoring of SEQUEST Search Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 78
Viewing the SEQUEST Search Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79
Viewing the Multiple SEQUEST Search Results. . . . . . . . . . . . . . . . . . . . . . . . 80
Viewing Protein Comparison Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 84
Contents
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Interpreting the SEQUEST Search Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85
DTA Information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 86
The Chromatogram. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87
The Results Table . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 88
Results Views. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 98
SEQUEST Results Summary View . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 109
Viewing Summary Results. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 109
Protein Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 109
Loading Protein Results. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 110
The Protein Results Report . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 110
Filtering and Sorting the Search Results . . . . . . . . . . . . . . . . . . . . . . . . . . . 112
Chapter 5 PepQuan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .117
About PepQuan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 118
ICAT/c-ICAT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119
iTRAQ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 122
SILAC. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 127
Peak Area/Height . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 132
Manual Integration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 135
Chapter 6 Exporting and Reporting Data. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .137
Protein and Peptide Reports . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 137
Data Exporting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 141
Exporting Data From the SEQUEST Results Table . . . . . . . . . . . . . . . . . . . . 143
Exporting a Chromatogram. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 143
Exporting Search Results to Microsoft Excel . . . . . . . . . . . . . . . . . . . . . . . . 143
Exporting Peptide Sequence Information to the Clipboard . . . . . . . . . . . . . 145
Exporting Search Results to an XML File . . . . . . . . . . . . . . . . . . . . . . . . . . 145
Exporting a DTA to PEAKS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 146
Copying DTA Files to a New Folder. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 147
Exporting Selected Proteins to a FASTA Database. . . . . . . . . . . . . . . . . . . . 147
Exporting Data From Other Results Views . . . . . . . . . . . . . . . . . . . . . . . . . . . 148
Exporting From the Display Ions View . . . . . . . . . . . . . . . . . . . . . . . . . . . . 148
Exporting From the Coverage View. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 150
Exporting Sequences from the Blast Results View . . . . . . . . . . . . . . . . . . . . 151
Exporting Protein Sequences from Retrieve Sequence View. . . . . . . . . . . . . 152
Exporting the Ion Table from the Sequence Ions View . . . . . . . . . . . . . . . . 153
Exporting from the Spectrum View. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 154
Pepmap and Pepmatch . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 155
Exporting from Pepmap . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 155
Exporting from Pepmatch . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 156
Contents
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Chapter 7 Working with Other BioWorks Tools . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .157
FASTA Database Tools . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 157
Indexing a FASTA Database . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 158
Database Utilities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 161
Reversing Database Entries in a FASTA Database . . . . . . . . . . . . . . . . . . . . 165
Fetching a Database. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 166
Find a FASTA Database Entries . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 170
FASTA and BLAST Databases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 172
Sequence Tools . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 173
Calculator . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 173
Sequence Comparison . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 175
Sequence Editor. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 176
Fragments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 178
Translating a Nucleotide Sequence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 182
Converting your Search Results. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 184
Converting SEQUEST Search Results. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 185
Converting Spectral Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 186
SEQUEST Queue Information. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 189
Other Software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 191
Cluster Manager . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 191
ProMass. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 191
Chapter 8 Pepmap and Pepmatch. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .193
About Pepmap . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 193
Using Pepmap. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 194
Opening the RAW Data File. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 194
Setting Up the Peptide/Protein Sequence . . . . . . . . . . . . . . . . . . . . . . . . . . 195
Setting the Static Modifications. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 197
Setting the Enzyme Digest Parameters. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 199
The Results Table . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 202
About Pepmatch . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 204
Using Pepmatch . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 204
Opening the RAW Data File . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 205
Setting Up the Peptide Sequence. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 206
Setting Static and Differential Modifications. . . . . . . . . . . . . . . . . . . . . . . . 207
Setting Fragmentation and General Parameters . . . . . . . . . . . . . . . . . . . . . . 208
The Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 209
Appendix A Reference . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .211
Amino Acid Mass Values. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 211
Modification Values . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 212
Enzyme Cleavage Properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 213
Fragment Ions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 214
Thermo Scientific BioWorks User Guide vii
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Preface
About This Guide
Welcome to BioWorks 3.3.1 SP1, a suite of layered software applications for protein
identification and quantification. With BioWorks 3.3.1 SP1, you can perform analyses of
mass spectrometry data from samples containing biological molecules such as proteins and
peptides.
Related Documentation
This guide includes information on procedures and parameters used in BioWorks 3.3.1 SP1.
Additional information can be found in the Help links within the software.
In addition to this guide, Thermo Scientific provides a .pdf version of the BioWorks 3.3.1
SP1 Installation Guide.
It is important to check the on-line product support website, www.thermo.com/finnigan, for
vital information regarding the life cycle of this product release.
Safety and Special Notices
Make sure you follow the precautionary statements presented in this guide. The safety and
other special notices appear in boxes.
Safety and special notices include the following:
CAUTION Highlights hazards to humans, property, or the environment. Each
CAUTION notice is accompanied by an appropriate CAUTION symbol.
IMPORTANT Highlights information necessary to prevent damage to software, loss of
data, or invalid test results; or may contain information that is critical for optimal
performance of the system.
Note Highlights information of general interest.
Preface
viii BioWorks User Guide Thermo Scientific
Contacting Us
There are several ways to contact Thermo Scientific.
Assistance
For new product updates, technical support, and ordering information, contact us in one of
the following ways:
Visit Thermo Scientific on the Web
www.thermo.com/finnigan
Contact Technical Support
Phone: 1-800-685-9535
Fax: 1-561-688-8736
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Tip Helpful information that can make a task easier.
Thermo Scientific BioWorks User Guide 1
1
Introduction
BioWorks is a configurable software package for peptide and protein mass spectrometry
analyses.
BioWorks features SEQUEST® protein search algorithm and PepQuan™ application for label
and label-free quantitative analysis of peptides. Use this suite of software applications to
analyze spectral data from all Thermo Fisher Scientific mass spectrometers.
This chapter contains the following sections:
•Understanding BioWorks
•What is New in BioWorks
•Using BioWorks
Understanding BioWorks
BioWorks is used to provide protein identification from mass spectra of digested fragments.
BioWorks includes:
• SEQUEST search algorithm to identify proteins in complex mixtures.
• Interactive navigation tools to filter and sort protein summaries and decrease the time
spent interpreting results.
• Customize and annotate spectral plots and chromatograms for professional publications
and presentations by using the PEPMATCH™ and PEPMAP™ tools.
• Batch processing capability to improve throughput by queuing up several files for batch
or overnight analysis.
• Unified search results file (SRF) to save all of the search parameter information and
results in a single binary file, thus simplifying and accelerating data access.
• Custom-build proprietary databases, index databases, and retrieve databases through a
public server.
Database indexing creates sorted peptide fragment lists after in silico digestion protein or
nucleotide database sequences. The database index enables more rapid searches with
SEQUEST and faster identification of proteins.
1 Introduction
Understanding BioWorks
2BioWorks User Guide Thermo Scientific
BioWorks also allows post-translational modifications to be added after an indexed
database has been created. With this version of software there is no need to re-index each
time a new modification is introduced.
Combining the traditional sensitivity of SEQUEST with the stringency of a probability-based
scoring algorithm, BioWorks provides the flexibility to complete results and improve
confidence in your peptide and protein matches.
This sections describes the following features of BioWorks:
•SEQUEST Search Algorithm
•PepQuan
•Results Interpretation Exporting and Reporting Data
•Pepmap
•Pepmatch
•Additional Databases and Sequence Tools
SEQUEST Search Algorithm
SEQUEST uses the high-level specificity of tandem mass spectra to identify proteins by
correlating peptide MS/MS data with in silico fragmentation patterns predicted from protein
or nucleotide databases. Use SEQUEST in combination with automated LC/MS/MS and
intelligent data acquisition tools to ensure the identification of low abundance proteins in
complex mixtures is routine.
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 are low.
The practical, interactive data summary allows information to be extracted. It only takes a
click to examine a fully annotated MS/MS spectrum, upload a sequence to NCBI for searches
of FASTA and BLAST Databases, 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. In addition, SEQUEST optimizes for accurate mass searching on data
acquired with Thermo Scientific LTQ FT and LTQ Orbitrap hybrid mass spectrometers.
Default search parameters are set in parts per million (ppm) depending on the instrument
used for data acquisition and there are additional features for searching with narrow mass
windows. Use of accurate mass windows decreases the time of the search and increases the
accuracy of the result.
1 Introduction
Understanding BioWorks
Thermo Scientific BioWorks User Guide 3
The BioWorks probability-based scoring system rates the relevance of the best matches found
by the SEQUEST algorithm. This probability-based scoring allows BioWorks to
independently rank the peptides and proteins and to increase the confidence in protein
identification. Additionally, it minimizes the time needed for data interrogation or results
review, increasing the overall throughput of the analysis.
False discovery rates can also be determined using reverse databases. BioWorks contains a tool
for easy creation of reverse databases. Comparison of the results with forward and reversed
databases provides an additional means of increasing confidence in protein identification.
For more information on how to use SEQUEST with BioWorks, refer to Chapter 3,
“Performing Search,” Chapter 4, “Viewing and Interpreting Search Results,” and Chapter 7,
“Working with Other BioWorks Tools.”
PepQuan
PepQuan assists in the quantification of isotopically labeled peptides by determining the ratio
of heavy to light ions using peak intensities. PepQuan’s proteomics method is designed to
measure changes in protein levels in cells. BioWorks can quantitate peptides with
MS/MS-based quantification approaches using chemical labels, such as iTRAQ™.
For more information about quantification, see Chapter 5, “PepQuan.”
Results Interpretation Exporting and Reporting Data
BioWorks offers extensive options for exporting data and results into other file formats such
as XML or PDF. You can use the reports to conduct detail analysis of your search results or to
share the results of your analysis.
To create reports you can use the available file formats XML, Text, Excel, and PDF. Define
your report parameters in the Configuration dialog box or through your Search Results dialog
box. For more information about exporting and reporting data, see Chapter 6, “Exporting
and Reporting Data.”
Pepmap
Use the Pepmap utility to predict the peptides resulting from an enzymatic digest of a protein
sequence. Pepmap can also match the predicted peptide fragments to observed masses in an
acquired mass spectrum.
For more information about Pepmap, see Chapter 8, “Pepmap and Pepmatch.”
1 Introduction
What is New in BioWorks
4BioWorks User Guide Thermo Scientific
Pepmatch
Use the Pepmatch utility to predict the ions produced by fragmentation of a particular
peptide sequence. Pepmatch can match the predicted fragment ions to observed masses in an
acquired MS/MS scan.
For more information about Pepmatch, see Chapter 8, “Pepmap and Pepmatch.”
Additional Databases and Sequence Tools
BioWorks includes a collection of database management and sequence tools that you can use
to download data, define searches, and edit, compare, and analyze sequences.
For more information about tools, see Chapter 7, “Working with Other BioWorks Tools.”
What is New in BioWorks
BioWorks 3.3.1 SP1 introduces the analysis of ETD (Electron Transfer Dissociation) spectra
using the SEQUEST search algorithm. ETD is an ion fragmentation technology that
dissociates peptides by transferring electrons to positively charged peptides. ETD
fragmentation tends to produce c and z ions. CID (Collision-Induced Dissociation)
fragmentation tends to produce b and y ions. The charge state range for ETD, compared to
CID, is greater. ETD and CID are complementary dissociation techniques. To achieve
greater coverage of a protein sequence use both ETD and CID analyses techniques.
This section describes the new features that help support the ETD analysis.
Activation Type
To analyze ETD spectra the activation type option was added to the SEQUEST Search dialog
box and to the SEQUEST Results and MultiConsensus Results reports.
Use the activation type in the SEQUEST Search dialog box to select ETD, CID, or PQD for
analysis. Using the information from this option, SEQUEST determines which data to
extract, analyze, and report.
BioWorks displays the activation type in the SEQUEST Results and MultiConsensus Results
reports. The search results can be filtered and sorted by the activation type. Additionally, CID
and ETD search results can be displayed in the same table for easy protein sequence analysis.
1 Introduction
Using BioWorks
Thermo Scientific BioWorks User Guide 5
Data Preprocessing
BioWorks 3.3.1 SP1 now performs data preprocessing of ETD spectra to determine precursor
ion charge states from the RAW data file for efficient processing and searching.
ETD spectra are generated from highly charged peptides (MH+ >+3). The ETD ion-ion
reaction produces charge-reduced states of the precursor ions. These ions are normally
abundant signals in the ETD spectrum. The data preprocessing tool determines the charge
state of the original precursor ion for each ETD spectrum. Once the data preprocessing
option assigns the precursor ion charge state, this information is passed to the database search
algorithm for confident sequence determination.
In some cases (such as very large peptides), the reduced precursor charge states are outside the
scanning mass range of the ETD tandem mass spectrum. In these cases, the confident
assignments of the precursor charge states are not possible. In this situation, the data
preprocessing determines two possible charge states. For example, +2 and +3, +2 and + 6, +3
and +6, +4 and +6, or +5 and +6.
Using BioWorks
BioWorks processes data from protein and peptide LC/MS analysis. Large proteins are
subjected to enzymatic digestion prior to LC/MS analysis to yield mixtures of peptides. The
mixture of peptides is subjected to LC/MS/MS analysis to separate the peptides by
chromatography and then produce MS/MS spectra of the individual peptides. The MS/MS
spectra can be generated either with Collision-Induced Dissociation (CID) or with Electron
Transfer Dissociation (ETD). The peptide fragments generated by these two dissociation
processes are different and can provide complementary information.
BioWorks searches through the resulting MS/MS spectra of peptides to match the peptide
spectra against theoretical peptide spectra derived from a protein sequence in a database.
BioWorks’ correlation and statistical analysis determines how exact the match is between the
theoretical spectrum and the experimental spectrum. BioWorks takes the confidently assigned
peptide sequences and assembles a list of proteins. It calculates the protein sequences coverage
of the list of proteins.
1 Introduction
Using BioWorks
6BioWorks User Guide Thermo Scientific
BioWorks Work Flow
To use BioWorks to do a SEQUEST analysis, complete six basic steps
1. Load a .raw file.
Use a peptide sample that was generated from a protein digestion and has been analyzed
either by LC-MS/MS or MALDI-MS/MS.
2. Select the SEQUEST search engine and set four required SEQUEST parameters. See
“Setting Basic SEQUEST Search Options” on page 48.
3. Select and modify the DTA generation settings. See “Modifying the DTA Generation
Parameters” on page 54.
4. Define your search parameter settings. See “Setting the SEQUEST Search Parameters”
on page 56.
Create a search parameters file that contains information about the instrument, possible
modifications, and the database to be used.
5. Start your SEQUEST search and save results.
6. View and compare multiple SEQUEST search results, use MultiConsensus Results view
or Load Protein Comparisons view. See “Viewing the Multiple SEQUEST Search
Results” on page 80 or “Viewing Protein Comparison Results” on page 84.
1 Introduction
Using BioWorks
Thermo Scientific BioWorks User Guide 7
Figure 1. BioWorks with SEQUEST Work Flow
Load a Raw file
Analyze results in MultiConsensus
Start search and Save results
Specify parameters
(C Ions, Y Ions, Z Ions)
Select SEQUEST search engine
DTA generation setting
(ETD preprocessing)
Set basic parameters
- Define SRF file
- Select instrument type
- Define scan range
- Select activation type
ETD activation type CID activation type
Select and modify
DTA generation setting
Specify parameters
(B Ions and Y Ions)
1 Introduction
Using BioWorks
8BioWorks User Guide Thermo Scientific
BioWorks Input and Output File Types
BioWorks accepts and creates the file types listed in Table 1 .
Unified Search File
BioWorks contains a Unified Search File format that creates a single file called a .srf file. The
.srf file contains relevant information from three separate sources: information used to set up
the search, .dta file data, and .out files data.
Choose File > Open Search Results File to open the Unified Search File.
The Open MultiConsensus Results report and Open Protein Expression Results dialog boxes
also support the Unified Search File. You can save all the data in one file from the search
results reports in the .srf files.
Raw File
A .raw file is a data file generated from and analyzed by either LC-MS/MS or
MALDI-MS/MS system. It contains experiment information, parameter settings, sample
information, mass value data, and fragmentation process information.
Table 1. File Types
File Extension Content Description File Type
.raw raw data raw data collected from an
instrument
input
.srf Unified Search File contains relevant
information from three
separate sources
input and
output
.dta mass spectral files produced
during SEQUEST analysis
contains MSn data for single
or grouped scans
input and
output
.out output BioWorks files contains the search results
for one .dta file
output
.sld Xcalibur sequence file a complete set of vMALDI
data
input
Thermo Scientific BioWorks User Guide 9
2
Setting your Default Configuration
This chapter describes how to define search parameters, configure the global default settings,
and define output results. Use the Configuration options to set these initial default values for
the BioWorks Browser before you begin using the search and analysis tools.
This chapter contains the following sections:
•About Configuration
•Setting Default Parameters
•General Configuration Options
•Search Engine Configuration Options
•Results Configuration Options
•PepQuan Configuration Options
2 Setting your Default Configuration
About Configuration
10 BioWorks User Guide Thermo Scientific
About Configuration
The configuration dialog box is divided into four categories:
•General
•Search Engines
•Results
•PepQuan
You can set your BioWorks defaults values in these configuration pages. You can also choose
to use the original BioWorks configuration default values.
If you are a first time user, you must set the defaults for SEQUEST Cluster, SEQUEST
Sorcerer and Mascot Search.
Table 2. Configuration Pages
Category Configuration Page
General
Default Folder
Internet URLs
Mass Options
Tr a c e O p t i o n s
Enzymes
Modifications Menu
Static Modifications
Search Engines
DTA Generation
DTA Filters
FASTA Database Indexer
SEQUEST Search
SEQUEST Cluster
SEQUEST Sorcerer
Mascot Search
Results
Results Table
Search Results
Results Filters
2 Setting your Default Configuration
Setting Default Parameters
Thermo Scientific BioWorks User Guide 11
Setting Default Parameters
To set default parameters in the Configuration pages
1. Choose Options > Configuration to open the Configuration page.
2. Click one of the configuration pages in the left panel. The default parameters of the
selected page appear in the right panel.
3. Set the parameters in the fields in the right panel.
4. Click Apply.
5. Click OK to save the changes for the active page and close the box.
All Configuration pages are listed in the left panel of the Configuration box. When you click
a Configuration page, the default values for that page appear in text boxes in the right panel.
See Figure 2.
Change the values in the text boxes to configure the page you have selected. Many settings can
also be modified with the individual analysis tools before performing the analysis.
This section describes each Configuration page in detail.
Display Ions
PepQuan
Isotope Labeling Reagents
ICAT/c-ICAT
PepQuan iTRAQ
PepQuan SILAC
Peak Area/Height
ICIS Peak Detection
Table 2. Configuration Pages
Category Configuration Page
Note To restore the installationt default values, click Defaults. The Defaults button
is not available when the Default Folders page is active.
2 Setting your Default Configuration
General Configuration Options
12 BioWorks User Guide Thermo Scientific
General Configuration Options
Use the Configuration page to set general options, such as the default search folder, Internet
URLs, mass options, trace options, enzymes, and modifications.
Default Folder
To set defaults for SEQUEST searches, click Default Folder in the left panel, as shown in
Figure 2.
Figure 2. Default Folders page
Table 3 describes the parameters in the Default Folders page.
Table 3. Default Folder parameters
Parameter Functions
RAW Files Specifies the location of the .raw files.
FASTA Database Specifies the location of the FASTA databases.
Protein/Peptide Files Specifies the location for the protein and peptide sequence files
(.pep) used by Pepmatch and Pepmap.
SEQUEST Results Specifies the location for the SEQUEST search results files (.srf,
.dta, and .out files).
SEQUEST Params Specifies the location for the SEQUEST parameters file (.params).
/