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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
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
• 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.
/
