Thermo Fisher Scientific BioWorks 3.3.1 SP1 User guide

For Research Use Only

Not for use in Diagnostic Procedures

XCALI-97194 Revision B May 2007

Xcalibur

BioWorks

User Guide

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

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

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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

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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).

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Thermo Fisher Scientific BioWorks 3.3.1 SP1 User guide

Thermo Fisher Scientific BioWorks 3.3.1 SP1 User guide

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