Thermo Fisher Scientific Proteome Discoverer 1.1 User guide

Xcalibur

Proteome Discoverer

Version 1.1

User Guide

XCALI-97276 Revision A October 2009

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

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

Contents

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

Contents

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

Contents

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.

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Thermo Fisher Scientific Proteome Discoverer 1.1 User guide

Thermo Fisher Scientific Proteome Discoverer 1.1 User guide

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