Thermo Fisher Scientific BioPharma Finder 1.0 User guide

Thermo

BioPharma Finder

User Guide

Software Version 1.0

XCALI-97791 Revision A December 2015

Exactive Plus and BioPharma Finder are trademarks, and Exactive, LCQ, LTQ, LTQ FT, Orbitrap, Thermo

Scientific, and Xcalibur are registered trademarks of Thermo Fisher Scientific Inc. in the United States.

The following are registered trademarks in the United States and other countries: Excel, Microsoft, Internet

Explorer, PowerPoint, and Windows are registered trademarks of Microsoft Corporation. Adobe, Acrobat, and

Reader are registered trademarks of Adobe Systems Incorporated.

Mascot is a registered service mark of Matrix Science Ltd. in the United States and possibly other countries.

ReSpect is a trademark of Positive Probability Ltd.

All other trademarks are the property of Thermo Fisher Scientific Inc. and it 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.

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, December 2015

Software version: Thermo BioPharma Finder 1.0 and later, Microsoft Windows 7 Professional SP1 (64-bit)

For Research Use Only. Not for use in diagnostic procedures.

Thermo Scientific BioPharma Finder User Guide iii

C

Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix

Related Documentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .ix

System Requirements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .xi

License Activation and Deactivation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .xi

Special Notices . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xii

Contacting Us . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xii

Chapter 1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1

BioPharma Finder Features . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1

Protein Sequence Manager . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2

Peptide Mapping Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3

Intact Protein Analysis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9

Starting the BioPharma Finder Application. . . . . . . . . . . . . . . . . . . . . . . . . . . . 19

Setting Up Peptide Mapping Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20

Setting Up Intact Protein Analysis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21

Specifying the Default Raw Data File Directory . . . . . . . . . . . . . . . . . . . . . . 22

Specifying the Precision for Output Mass Values. . . . . . . . . . . . . . . . . . . . . . 22

Exiting the BioPharma Finder Application . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23

Chapter 2 Using the Protein Sequence Manager and Editor. . . . . . . . . . . . . . . . . . . . . . . . . .25

Creating and Editing Protein Sequences . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25

Protein Sequence Manager Page Parameters . . . . . . . . . . . . . . . . . . . . . . . . . 31

Protein Sequence Editor Parameters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32

Matching Target Sequence Components. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34

Modifying Protein Sequences . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35

Changing the Default Modifications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35

Applying Modifications to a Protein Sequence . . . . . . . . . . . . . . . . . . . . . . . 36

Creating Custom Modifications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48

Saving the Protein Sequence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52

Contents

iv BioPharma Finder User Guide Thermo Scientific

Chapter 3 Performing a Peptide Mapping Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .55

Data Acquisition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55

Creating and Processing a New Experiment . . . . . . . . . . . . . . . . . . . . . . . . . . . 55

Loading the Raw Data Files. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58

Selecting a Sequence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60

Selecting or Deleting a Method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61

Creating or Editing a Processing Method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63

Editing Data Processing Parameters. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64

Saving the Method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81

Managing the Run Queue . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83

Running the Jobs. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 84

Queue Page Parameters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87

Parameters Page Parameters. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89

Chapter 4 Viewing the Peptide Mapping Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .101

Viewing the Process and Review Page . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101

Opening the Results from the Queue Page . . . . . . . . . . . . . . . . . . . . . . . . . 103

Opening the Results from the Load Results Page. . . . . . . . . . . . . . . . . . . . . 104

Viewing the Results Table . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 106

Viewing the Chromatograms. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 108

Viewing the Fragment Coverage Map . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 113

Viewing the Protein Sequence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 115

Viewing the Deconvolved and Full-Scan MS Spectra . . . . . . . . . . . . . . . . . 116

Viewing the Predicted and Experimental MS/MS Spectra . . . . . . . . . . . . . . 117

Performing the Kinetic MS/MS Model Prediction . . . . . . . . . . . . . . . . . . . 124

Identifying Components Using De Novo Sequencing . . . . . . . . . . . . . . . . . 127

Reprocessing Results Using Real-Time Optimization . . . . . . . . . . . . . . . . . 131

Process and Review Page Parameters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 133

Process and Review Page Command . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 143

Process and Review Page Shortcut Menus . . . . . . . . . . . . . . . . . . . . . . . . . . 143

Viewing the Coverage Page . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 145

Viewing the Protein Coverage Results Table . . . . . . . . . . . . . . . . . . . . . . . . 146

Viewing the Color-Coded Chromatograms . . . . . . . . . . . . . . . . . . . . . . . . . 147

Viewing the Protein Coverage Map . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 151

Coverage Page Parameters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 153

Coverage Page Shortcut Menus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 155

Viewing the Modification Summary Page . . . . . . . . . . . . . . . . . . . . . . . . . . . . 156

Viewing the Modification Results Pane . . . . . . . . . . . . . . . . . . . . . . . . . . . . 158

Viewing the Components Table . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 162

Modification Summary Page Parameters . . . . . . . . . . . . . . . . . . . . . . . . . . . 164

Modification Results Pane Parameters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 166

Modification Summary Page Shortcut Menus . . . . . . . . . . . . . . . . . . . . . . . 169

Contents

Thermo Scientific BioPharma Finder User Guide v

Chapter 5 Manually Deconvolving Isotopically Resolved Mass Spectra with the

Xtract Algorithm . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .171

Setting Up a Manual Xtract Protein Deconvolution . . . . . . . . . . . . . . . . . . . . 172

Intact Protein Analysis Page Parameters for the Xtract Algorithm . . . . . . . . 175

Creating a Method for Xtract Deconvolution . . . . . . . . . . . . . . . . . . . . . . . . . 176

Creating a Method for a Single Xtract Deconvolution. . . . . . . . . . . . . . . . . 177

Creating a Method for an Xtract Sliding Window Deconvolution. . . . . . . . 184

Creating a Method for Xtract Target Sequence Matching . . . . . . . . . . . . . . 189

Creating a Report for Xtract Deconvolution . . . . . . . . . . . . . . . . . . . . . . . . . . 195

Parameters Page Parameters for the Xtract Algorithm . . . . . . . . . . . . . . . . . 198

Menu Bar Commands on the Parameters Page . . . . . . . . . . . . . . . . . . . . . . 217

Selecting the Spectrum to Deconvolve for Xtract. . . . . . . . . . . . . . . . . . . . . . . 217

Selecting the Spectrum to Deconvolve for a Single Xtract

Deconvolution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 218

Selecting the Parameters for an Xtract Sliding Window Deconvolution. . . . 225

Selecting the Spectrum to Deconvolve for Xtract Target Sequence

Matching . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 227

Chromatogram Page Parameters for the Xtract Algorithm. . . . . . . . . . . . . . 228

Chromatogram Page Menu Bar Commands . . . . . . . . . . . . . . . . . . . . . . . . 229

Chromatogram Page Shortcut Menus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 229

Chromatogram Pane Header . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 230

Source Spectrum Pane Shortcut Menu . . . . . . . . . . . . . . . . . . . . . . . . . . . . 230

Source Spectrum Pane Header. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 230

Deconvolving the Spectrum with Xtract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 231

Deconvolving the Spectrum for a Single Xtract Deconvolution. . . . . . . . . . 231

Deconvolving the Spectrum for an Xtract Sliding Window

Deconvolution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 233

Deconvolving the Spectrum for Xtract Target Sequence Matching . . . . . . . 236

Displaying the Results for Xtract Deconvolution. . . . . . . . . . . . . . . . . . . . . . . 236

Displaying the Results of a Single Xtract Deconvolution . . . . . . . . . . . . . . . 237

Displaying the Results of an Xtract Sliding Window Deconvolution. . . . . . 243

Displaying the Results of an Xtract Target Sequence Matching

Deconvolution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 246

Selecting a Reference Mass to Calculate Mass Differences in Xtract. . . . . . . 248

Adjusting the Xtract Deconvolution Results . . . . . . . . . . . . . . . . . . . . . . . . 249

Process and Review Page Parameters for the Xtract Algorithm. . . . . . . . . . . 249

Process and Review Page Menu Bar Commands . . . . . . . . . . . . . . . . . . . . . 254

Process and Review Page Shortcut Menus . . . . . . . . . . . . . . . . . . . . . . . . . . 255

Comparing Samples in Xtract Deconvolution . . . . . . . . . . . . . . . . . . . . . . . . . 256

Comparing Samples in Saved Xtract Results . . . . . . . . . . . . . . . . . . . . . . . . 268

Sample Comparison Page Parameters for the Xtract Algorithm . . . . . . . . . . 268

Sample Comparison Page Menu Bar Commands . . . . . . . . . . . . . . . . . . . . 270

Mirror Plot Shortcut Menu . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 271

Contents

vi BioPharma Finder User Guide Thermo Scientific

Displaying an Xtract Deconvolution Report . . . . . . . . . . . . . . . . . . . . . . . . . . 271

Reporting Page Toolbar. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 274

Sample Information Section . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 275

Chromatogram Parameters Section . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 276

Source Chromatogram Section . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 277

Main Parameters (Xtract) Section . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 278

Advanced Parameters (Xtract) Section . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 278

Sliding Window Parameters Section . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 279

Source Spectrum Section . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 279

Deconvolved Spectrum Section . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 280

Mirror Plot Section . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 281

Xtract Masses Table Section . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 282

Source Spectrum Evidence Section . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 283

Saving the Results of an Xtract Deconvolution . . . . . . . . . . . . . . . . . . . . . . . . 286

Exporting the Results of an Xtract Deconvolution. . . . . . . . . . . . . . . . . . . . . . 287

Deleting the Results of an Xtract Deconvolution. . . . . . . . . . . . . . . . . . . . . . . 289

Loading Saved Xtract Results. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 290

Chapter 6 Automatically Deconvolving Isotopically Resolved Mass Spectra with the

Xtract Algorithm . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .293

Setting Up an Automatic Xtract Deconvolution . . . . . . . . . . . . . . . . . . . . . . . 293

Creating a Method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 297

Running Jobs in the Queue. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 298

Queue Page Parameters for Automatic Xtract Deconvolution . . . . . . . . . . . 302

Comparing Samples Generated with the Automatic Xtract Algorithm. . . . . . . 304

Displaying the Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 305

Result Names . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 306

Displaying an Xtract Deconvolution Report . . . . . . . . . . . . . . . . . . . . . . . . . . 307

Saving an Xtract Deconvolution Report . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 308

Loading Saved Xtract Results. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 308

Chapter 7 Manually Deconvolving Isotopically Unresolved Mass Spectra with the

ReSpect Algorithm . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .309

Setting Up a Manual ReSpect Protein Deconvolution. . . . . . . . . . . . . . . . . . . 311

Intact Protein Analysis Page Parameters for the ReSpect Algorithm. . . . . . . 314

Creating a Method for ReSpect Deconvolution. . . . . . . . . . . . . . . . . . . . . . . . 316

Creating a Method for a Single ReSpect Deconvolution . . . . . . . . . . . . . . . 317

Creating a Method for a ReSpect Sliding Window Deconvolution . . . . . . . 326

Creating a Method for ReSpect Target Sequence Matching. . . . . . . . . . . . . 332

Creating a Method for Deconvolution with Ion Trap Data. . . . . . . . . . . . . 339

Creating a Method for the Structural Analysis of Proteins . . . . . . . . . . . . . . 339

Creating a Report for ReSpect Deconvolution. . . . . . . . . . . . . . . . . . . . . . . . . 339

Parameters Page Parameters for the ReSpect Algorithm. . . . . . . . . . . . . . . . 343

Menu Bar Commands on the Parameters Page . . . . . . . . . . . . . . . . . . . . . . 367

Contents

Thermo Scientific BioPharma Finder User Guide vii

Selecting the Spectrum to Deconvolve for ReSpect . . . . . . . . . . . . . . . . . . . . . 367

Selecting the Spectrum to Deconvolve for a Single ReSpect

Deconvolution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 368

Obtaining the Best Results with the ReSpect Algorithm . . . . . . . . . . . . . . . 375

Selecting the Parameters for a ReSpect Sliding Window Deconvolution . . . 375

Selecting the Spectrum to Deconvolve for ReSpect Target Sequence

Matching . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 377

Chromatogram Page Parameters for the ReSpect Algorithm . . . . . . . . . . . . 378

Chromatogram Page Menu Bar Commands . . . . . . . . . . . . . . . . . . . . . . . . 379

Chromatogram Pane Shortcut Menus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 379

Chromatogram Pane Header . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 380

Source Spectrum Pane Shortcut Menu . . . . . . . . . . . . . . . . . . . . . . . . . . . . 380

Source Spectrum Pane Header. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 380

Deconvolving the Spectrum with ReSpect. . . . . . . . . . . . . . . . . . . . . . . . . . . . 381

Deconvolving the Spectrum for a Single ReSpect Deconvolution . . . . . . . . 381

Deconvolving the Spectrum for a ReSpect Sliding Window

Deconvolution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 383

Deconvolving the Spectrum for Target Sequence Matching . . . . . . . . . . . . 386

Displaying the Results for ReSpect Deconvolution . . . . . . . . . . . . . . . . . . . . . 386

Displaying the Results of a Single ReSpect Deconvolution . . . . . . . . . . . . . 387

Displaying the Results of a ReSpect Sliding Window Deconvolution . . . . . 393

Displaying the Results of a ReSpect Target Sequence Matching

Deconvolution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 397

Selecting a Reference Mass to Calculate Mass Differences in ReSpect . . . . . 400

Calculating a Protein Quality Score. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 400

Specifying the Output Mass Range . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 402

Adjusting the ReSpect Deconvolution Results . . . . . . . . . . . . . . . . . . . . . . . 403

Process and Review Page Parameters for the ReSpect Algorithm . . . . . . . . . 403

Process and Review Page Menu Bar Commands . . . . . . . . . . . . . . . . . . . . . 408

Process and Review Page Shortcut Menus . . . . . . . . . . . . . . . . . . . . . . . . . . 409

Comparing Samples in ReSpect Deconvolution . . . . . . . . . . . . . . . . . . . . . . . 410

Comparing Samples in Saved ReSpect Results. . . . . . . . . . . . . . . . . . . . . . . 422

Sample Comparison Page Parameters for the ReSpect Algorithm . . . . . . . . 422

Sample Comparison Page Menu Bar Commands . . . . . . . . . . . . . . . . . . . . 425

Mirror Plot Shortcut Menu . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 425

Contents

viii BioPharma Finder User Guide Thermo Scientific

Displaying a ReSpect Deconvolution Report. . . . . . . . . . . . . . . . . . . . . . . . . . 425

Reporting Page Toolbar. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 428

Sample Information Section . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 428

Chromatogram Parameters Section . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 430

Source Chromatogram Section . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 430

Main Parameters (ReSpect) Section. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 432

Advanced Parameters (ReSpect) Section . . . . . . . . . . . . . . . . . . . . . . . . . . . 432

Sliding Window Parameters Section . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 432

Source Spectrum Section . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 433

Deconvolved Spectrum Section . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 434

Mirror Plot Section . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 435

ReSpect Masses Table Section . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 436

Source Spectrum Evidence Section . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 437

Saving the Results of a ReSpect Deconvolution . . . . . . . . . . . . . . . . . . . . . . . . 440

Exporting the Results of a ReSpect Deconvolution . . . . . . . . . . . . . . . . . . . . . 441

Deleting the Results of a ReSpect Deconvolution . . . . . . . . . . . . . . . . . . . . . . 443

Loading Saved ReSpect Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 444

Chapter 8 Automatically Deconvolving Isotopically Unresolved Mass Spectra with

the ReSpect Algorithm . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .447

Setting Up an Automatic ReSpect Deconvolution. . . . . . . . . . . . . . . . . . . . . . 448

Creating a Method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 452

Running Jobs in the Queue. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 454

Queue Page Parameters for Automatic ReSpect Deconvolution. . . . . . . . . . 457

Comparing Samples Generated with the Automatic ReSpect Algorithm . . . . . 459

Displaying the Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 460

Result Names . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 461

Displaying a ReSpect Deconvolution Report. . . . . . . . . . . . . . . . . . . . . . . . . . 462

Saving an Xtract Deconvolution Report . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 463

Loading Saved Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 463

Appendix A Interactive Functions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .465

Rearranging the Panes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 465

Repositioning the Panes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 465

Resizing the Panes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 468

Using Basic Chromatogram Functions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 471

Using Basic Spectrum Functions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 472

Using the Copy and Paste Functions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 472

Using Basic Table Functions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 474

Filtering Data in a Table . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 476

Appendix B N-Linked Glycans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .483

Appendix C References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .495

Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .497

Thermo Scientific BioPharma Finder User Guide ix

P

Preface

This guide describes how to use the Thermo BioPharma Finder™ 1.0 application to

characterize biotherapeutic proteins by using two key workflows: peptide mapping analysis

and intact protein analysis.

To suggest changes to the documentation or to the Help

Complete a brief survey about this document by clicking the button below.

Thank you in advance for your help.

Related Documentation

The BioPharma Finder application includes complete documentation, including the Help

and the following documents as PDF files:

•Thermo BioPharma Finder Quick Start Guide briefly describes how to use the BioPharma

Finder application to analyze biotherapeutic protein experiments and identify peptides.

•Thermo BioPharma Finder User Guide describes how to use the main features of the

BioPharma Finder application.

Contents

•Related Documentation

•System Requirements

•License Activation and Deactivation

•Special Notices

•Contacting Us

Preface

xBioPharma Finder User Guide Thermo Scientific

To view the product manuals

• From the Microsoft™ Windows™ taskbar, choose Start > All Programs > Thermo

BioPharma Finder > Manuals > manual name.

• From the BioPharma Finder window, choose Help > Manuals > manual name.

To view the BioPharma Finder Help

• From the application window, choose Help > BioPharma Finder Help. To locate a

particular topic, use the Help Contents, Index, or Search panes.

• If information is available for a specific view, page, or dialog box, click Help or press the

F1 key to view this information.

• In applications that have a Communicator bar, click the field or parameter to display

definitions, required actions, ranges, defaults, and warnings, if they are available.

To download user documentation from the Thermo Scientific™ website

1. Go to www.thermoscientific.com.

2. In the Search box, type the product name and press ENTER.

3. In the left pane, select Documents & Videos, and then under Refine By Category, click

Operations and Maintenance.

4. (Optional) Narrow the search results or modify the display as applicable:

• For all related user manuals and quick references, click Operator Manuals.

• For installation and preinstallation requirements guides, click Installation

Instructions.

• For documents translated into a specific language, use the Refine By Language

feature.

• Use the Sort By options or the Refine Your Search box (above the search results

display).

5. Download the document as follows:

a. Click the document title or click Download to open the file.

b. Save the file.

Preface

Thermo Scientific BioPharma Finder User Guide xi

System Requirements

The following are the minimum and recommended system requirements for BioPharma

Finder 1.0 operation.

License Activation and Deactivation

Use the Thermo Scientific Product Licensing wizard to activate or deactivate the license for

the BioPharma Finder application. To activate the license, you must have an activation code

from Thermo Fisher Scientific. You must deactivate the license before you transfer it to

another computer.

To start the license activation or deactivation process

1. Open the BioPharma Finder application.

2. Choose Help > About BioPharma Finder to display the About dialog box.

3. Click Activate (Deactivate) to start the activation or deactivation process, as applicable.

4. Follow the instructions in the License Activation wizard.

For additional instructions, click Help in the wizard.

System Minimum requirements Recommended requirements

Hardware • Intel™ Core™ i7-4770

[email protected] GHz

• 8 GB registered RAM

• 916 GB storage hard drive—

ST1000DM-003 SCSI disk

device

• Resolution display 1280 1024

(XGA)

•DVD/CD-ROM drives

• Quad-core Intel Xeon CPU

(E5-1630 v3 3.7 GHz 10 MB

2133 4C)

• 32 GB DDR4-2133 (4 8 GB)

registered RAM

• 512 GB solid state boot drive

(SATA)

• 2 TB storage hard drive (SATA,

7200 rpm)

•DVD-RW optical drive

• Resolution display 1920 1080

Software • Microsoft™ Windows™ 7

Professional (English) SP1

(64-bit)

• Microsoft .NET Framework

4.5.1

• Microsoft Office 2010

• Adobe™ Reader™ 11

• Adobe™ Acrobat™ Pro

• Microsoft Windows 7

Professional (English) SP1

(64-bit)

• Microsoft .NET Framework

4.5.1

• Microsoft Office 2013

•Adobe Reader 11

•Adobe Acrobat Pro

Preface

xii BioPharma Finder User Guide Thermo Scientific

Special Notices

This guide uses the following types of special notices.

Contacting Us

There are several ways to contact Thermo Fisher Scientific for the information you need. You

can use your smartphone to scan a QR code, which opens your email application or browser.

IMPORTANT Depending on the features that you purchased in the BioPharma Finder

software, you have three licensing options:

• Option 1: Full license for all features in the application

• Option 2: Peptide Mapping Analysis features only

• Option 3: Intact Protein Analysis features only

The license keys control the different features in the software and determine which ones

are active. For example, if your purchase includes only Peptide Mapping Analysis, only the

Protein Sequence Manager and Peptide Mapping Analysis features are active when you

apply the license key. The Intact Protein Analysis feature is inactive. You can purchase the

additional features at a later date.

If you have a demonstration license key, when it expires, you must obtain a permanent

license key to continue using the BioPharma Finder application.

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.

Contact us Customer Service and Sales Technical Support

(U.S.) 1 (800) 532-4752 (U.S.) 1 (800) 532-4752

(U.S.) 1 (561) 688-8731 (U.S.) 1 (561) 688-8736

Preface

Thermo Scientific BioPharma Finder User Guide xiii

us.customer-support.analyze

@thermofisher.com

us.techsupport.analyze

@thermofisher.com

To find global contact information or customize your request

1. Go to www.thermoscientific.com.

2. Click Contact Us, select the Using/Servicing a Product option, and then

type the product name.

3. Use the phone number, email address, or online form.

To find product support, knowledge bases, and resources

Go to www.thermoscientific.com/support.

To find product information

Go to www.thermoscientific.com/lc-ms.

Note To provide feedback for this document:

• Send an email message to Technical Publications (techpubs-lcms@thermofisher.com).

• Complete a survey at www.surveymonkey.com/s/PQM6P62.

Contact us Customer Service and Sales Technical Support

Preface

xiv BioPharma Finder User Guide Thermo Scientific

Thermo Scientific BioPharma Finder User Guide 1

1

Introduction

The following topics describe the features and workflow of the BioPharma Finder application,

and explain how to start and exit the application.

BioPharma Finder Features

The BioPharma Finder application is a powerful tool for in-depth characterization of

biotherapeutic proteins. It automates the analysis of LC/MS for intact mass analysis and

LC/MS/MS data for peptide mapping to provide identity and relative quantitation of

proteins, variants, and low-level post translational modifications (PTMs).

You can use this application to analyze the smaller, larger, or even complex intact proteins

with mass deconvolution algorithms. You can also use this application to identify peptides

from the proteolytic digestion of a recombinant or natural protein with peptide fingerprinting

techniques.

The BioPharma Finder application consolidates the Thermo Scientific PepFinder and Protein

Deconvolution applications into one platform. It provides workflows for the top two key

analyses for protein characterization:

• Peptide Mapping Analysis

– Identifies peptides by using a new prediction algorithm (unique differentiator).

– Performs relative quantitation of post-translational modifications (PTMs).

Contents

•BioPharma Finder Features

•Starting the BioPharma Finder Application

•Setting Up Peptide Mapping Analysis

•Setting Up Intact Protein Analysis

•Exiting the BioPharma Finder Application

1 Introduction

BioPharma Finder Features

2BioPharma Finder User Guide Thermo Scientific

– Compares samples.

– Provides in-depth identification using error-tolerant search, amino acid substitution,

and de novo sequencing (unique differentiator).

• Intact Protein Analysis

– Provides molecular weight values of the biotherapeutic drugs and is necessary for

confirmation.

– Ensures high-quality results using two complementary deconvolution algorithms

optimized for isotopically resolved (Xtract) and unresolved data (ReSpect™). These

algorithms produce highly accurate results, even for low-abundance proteins, and

enable detection of extremely small protein modifications with mass shifts of just a

few daltons.

For more information about the primary features of the BioPharma Finder application, see

the following:

•Protein Sequence Manager

•Peptide Mapping Analysis

•Intact Protein Analysis

Protein Sequence Manager

The Protein Sequence Manager is the starting point for using the BioPharma Finder

application. It provides a central database of protein sequences so that you only have to load a

sequence once for Peptide Mapping Analysis or Intact Protein Analysis experiments.

Use the Protein Sequence Manager to do the following:

• Import a protein sequence from a FASTA file.

• Create a new sequence.

• Paste a sequence from a copied source.

• Define multiple chains—for example, two light chains and two heavy chains—for intact

analysis of unreduced antibodies, enabling the deconvolution of the ~150 kDa protein.

• Add fixed or variable modifications and disulfide bonds, as appropriate.

• Define a list of default modifications.

• Select a list of possible glycosylation structures.

For further information, see Chapter 2, “Using the Protein Sequence Manager and Editor.”

1 Introduction

BioPharma Finder Features

Thermo Scientific BioPharma Finder User Guide 3

Peptide Mapping Analysis

Peptide Mapping Analysis in the BioPharma Finder application analyzes a data-dependent

LC/MS/MS or full-scan LC/MS data from a proteolytic digestion of a few relatively pure

proteins, such as therapeutic proteins. It provides a high-confidence platform for fully

automated characterization of these proteins. Peptide Mapping Analysis performs component

detection, peptide identification, and identification and quantification of PTMs using

Thermo Scientific instruments. Figure 1 shows the Peptide Mapping Analysis page.

Figure 1. Peptide Mapping Analysis page

The following topics describe the main features of Peptide Mapping Analysis:

•Method Editor

•Run Queue

•Results Display

•Quantification of Modifications

•Deep Characterization Using Sequence Variant Analysis, Error-Tolerant Search, and De

Novo Sequencing

•Disulfide Mapping

•Localization of Glycosylation Sites on Glycopeptides

1 Introduction

BioPharma Finder Features

4BioPharma Finder User Guide Thermo Scientific

•Real-Time Optimization

•Peptide Mapping Analysis Workflow

Method Editor

A series of screens guides you through the different steps in creating a processing method,

including entering component detection and identification parameters, and then saving the

method. Included in the editor are interactive graphics for viewing the absolute MS signal

threshold. As you change this threshold, a red line that denotes it moves up and down

accordingly. The Method Editor in Figure 2 shows this line. You can view or save a summary

of the method settings on the last page before beginning processing.

For further information, see Creating or Editing a Processing Method.

Figure 2. Method Editor

Run Queue

Peptide Mapping Analysis features a run queue, where you can monitor an experiment’s

processing, stop jobs in progress, and open the results. You can enter a large number of

experiments at one time and let them run overnight to increase your productivity. While the

application processes experiments, you can simultaneously review results of other experiments

or perform Intact Protein Analysis data processing.

For further information, see Managing the Run Queue.

1 Introduction

BioPharma Finder Features

Thermo Scientific BioPharma Finder User Guide 5

Results Display

Peptide Mapping Analysis displays the results of a completed experiment in a Results table

that is organized by components at the upper level and by raw data files at the lower level. You

can export the data in the Results table to an Microsoft Excel™, a CSV, or an MGF file. The

Peptide Mapping Analysis results include the Modification Summary report, which shows the

recovery status and abundance of all detected modifications, and the Components table

displaying the components of a completed experiment related to a particular modification.

The application also displays the results in chromatograms, protein sequence maps, peptide

fragment coverage maps, protein sequences, full-scan spectra, and MS/MS spectra, including

predicted kinetic MS/MS spectra. You can view MS/MS spectra for each fragmentation type

and resolution combination.

The application generates both a base peak chromatogram (BPC) plot, which shows only the

most intense peak in each spectrum, and the selected ion chromatogram (SIC) plot, which

plots the intensity of the signal observed at a chosen m/z. Figure 3 shows these two types of

plots. You can view the base peak chromatogram in different shades of color to indicate

unidentified proteins and identified proteins by type.

Figure 3. BPC and SIC chromatograms

For further information, see Chapter 4, “Viewing the Peptide Mapping Results.”

1 Introduction

BioPharma Finder Features

6BioPharma Finder User Guide Thermo Scientific

Quantification of Modifications

You can use the Modification Summary report, shown in Figure 4, to see the amount of a

specific modification in the sample compared to the unmodified material. For example, if the

amino acid at position 32 in an antibody becomes oxidized as you create the molecule, you

must know how much of this oxidized amino acid is present in the sample and determine if

the level is too high. The BioPharma Finder application determines this level automatically.

Figure 4. Modification Summary report

Deep Characterization Using Sequence Variant Analysis, Error-Tolerant Search, and De Novo

Sequencing

Peptide Mapping Analysis offers a specific feature for detecting and characterizing

unanticipated sequence variants using the specialized amino acid substitution search. Not

only does the application identify low-level modification using MS/MS spectra, it also

automatically provides you with the abundance percentage in the sample. It achieves deeper

characterization by using an error-tolerant search to identify unknown modifications. It

searches user-definable mass ranges against identified peptides and easily identifies unexpected

modifications. You can use de novo sequencing to identify components that contain MS/MS

spectra that were not associated with the protein sequence imported into the processing

method. Through de novo sequencing, you can identify unexpected containments or

product-related impurities that can pose concerns regarding bioactivity, stability, and

immunogenicity of the biotherapeutics.

Page is loading ...

Thermo Fisher Scientific BioPharma Finder 1.0 User guide

Related papers

Other documents