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Preparing Samples
Voyager
™
Biospectrometry
™
Workstation User’s Guide 3-9
3
CHCA for thin layer
application
Use for peptides <3,000 Da.
Matrix concentration 20 mg/ml
Final sample concentration Up to 0.1 pmol/µl
Materials and solvents • Pure nitrocellulose (for example, Bio-Rad
Laboratories Trans-Blot
®
162-0146)
• Acetone
• Isopropanol
Preparation
1
1. Dissolve nitrocellulose to a concentration of
20 mg/ml in acetone by vortexing for 15 minutes.
2. Add isopropanol at a ratio of 1:1.
3. Add
α
-cyano-4-hydroxycinnamic acid to a final
concentration of 20 mg/ml.
Air dry sample plate after loading matrix. Dry with
gentle air flow after adding sample.
NOTE: If the dry matrix is a mustard-yellow color instead of bright yellow, it may
contain impurities. To recrystallize (purify), dissolve the
α
-cyano matrix in warm
ethanol. Filter and add about two volumes of deionized water. Let stand in the
refrigerator overnight. Filter, then wash the precipitate with cold water. This
procedure is not optimized for yield.
Crystals Rounded (see Figure 3-1 on page 3-34)
Stability Prepare weekly.
1. Other special sample preparation techniques are described in Shevchenko, A., M. Wilm, O. Vorm, M.
Mann,
Anal. Chem.,
1996, 68, 850–858.
- Biospectrometry 1
- Workstation 1
- Chapter 3 Preparing Samples 5
- ...................... 5-39 7
- ...................... 6-60 9
- Chapter 7 PSD Analysis 10
- Table of Contents 11
- Instrument Safety 13
- Safety symbols 15
- Electrical Symbols 15
- Non-electrical 16
- Symboles non 18
- Workstation User’s Guide xix 19
- Workstation User’s Guide xxi 21
- Safety and EMC 23
- Sécurité 24
- Laser Safety 25
- How to Use This Guide 27
- 1 Introducing the 31
- Workstations 31
- 1.1 Voyager 32
- -DE and 32
- Voyager-DE PRO 32
- System Overview 32
- Features 34
- 1.2 Voyager-DE STR 35
- 1.3 MALDI-TOF MS 37
- Technology Overview 37
- Ionization 38
- Workstation User’s Guide 1-9 39
- Advantages of 40
- MALDI-TOF 40
- 1.4 Voyager-DE 41
- Technology 41
- 1-12 PerSeptive Biosystems 42
- Extraction (Linear Mode) 43
- Benefits of 44
- Extraction 44
- Velocity focusing 45
- —Detection 46
- 1.5.1 System Components 47
- PB100465 47
- PB100466 48
- 1.5.2 Mass Spectrometer 50
- Parts of the mass 52
- 1.5.3 Vacuum System 54
- Vacuum pumps 56
- Vacuum gauges 56
- 1.5.4 Vacuum Gauge Panel 59
- 1-30 PerSeptive Biosystems 60
- 1.5.5 Computer Components 61
- 1.6.1 System Components 62
- 1.6.2 Mass Spectrometer 64
- 4113, and 4116 and later 66
- 1.6.3 Vacuum System 68
- (Top View) 69
- 1.6.4 Vacuum Gauge Panel 70
- 1.6.5 Front Panel Indicators 72
- 1.6.6 Computer Components 73
- Software 74
- 1.7 Software Overview 75
- 1-46 PerSeptive Biosystems 76
- (Data Explorer) 78
- 2 Installing the 79
- Voyager™ 79
- Biospectrometry™ 79
- 2.2.1 Voyager-DE and 80
- Voyager-DE PRO Workstations 80
- Selecting the Site 81
- Workstation User’s Guide 2-3 81
- PB100507 82
- 2-6 PerSeptive Biosystems 84
- 2-8 PerSeptive Biosystems 86
- PB100776 87
- PB100784 88
- 2.3.2 Connecting the 89
- 2.3.3 Connecting the 90
- 2.3.4 Connecting the 91
- LSA1000 LeCroy Digitizer 91
- 2-14 PerSeptive Biosystems 92
- PB100770 93
- 2-16 PerSeptive Biosystems 94
- NOTE: There are two 15-pin 96
- 2.4 Connecting the 97
- Voyager-DE STR Workstation 97
- Video monitors 100
- Biospectrometry 101
- 2.5 Installing Software 102
- Installing Software 103
- Accessing the 104
- 2.5.2 Starting the Software 105
- 2-28 PerSeptive Biosystems 106
- 2.5.3 Exiting the Software 107
- 2.6 Hardware Configuration 108
- Hardware Configuration 109
- 2-32 PerSeptive Biosystems 110
- High voltage 111
- Timed ion 112
- Instrument 113
- 2-36 PerSeptive Biosystems 114
- Digitizer 116
- In this section 117
- Overview 117
- When to align 117
- 2-40 PerSeptive Biosystems 118
- Alignment 119
- 2-42 PerSeptive Biosystems 120
- Using the control 121
- Before aligning 121
- Aligning 121
- 2-44 PerSeptive Biosystems 122
- Aligning the Sample Plate 123
- 2.8 Startup and Shutdown 124
- Startup and Shutdown 125
- 2-48 PerSeptive Biosystems 126
- Powering down 127
- 2.9 Checking System Status 128
- Checking System Status 129
- 2-52 PerSeptive Biosystems 130
- 3 Preparing Samples 131
- 3.1 Preparing Samples 132
- 3.1.1 Selecting a Matrix 133
- 3.1.2 Preparing Matrix 134
- Preparing Samples 135
- Workstation User’s Guide 3-5 135
- 3.1.3 Matrix Information 136
- Workstation User’s Guide 3-7 137
- 3-8 PerSeptive Biosystems 138
- Workstation User’s Guide 3-9 139
- 3-10 PerSeptive Biosystems 140
- DHB for neutral 142
- DHB for small 143
- 3-14 PerSeptive Biosystems 144
- Synthetic polymer 145
- 3-16 PerSeptive Biosystems 146
- 3.1.4 Preparing Sample 147
- Preparing 148
- Internal standards 148
- 3.1.5 Sample Cleanup 149
- 3.1.5.1 Washing 150
- When to use 151
- What you need 151
- Procedure 151
- 3-22 PerSeptive Biosystems 152
- 3.1.5.3 Cation Exchange 153
- 3.1.5.4 ZipTips 154
- (Dried Droplet Application) 156
- Mixing sample 157
- 3.2.1 Overview 158
- Types of sample 159
- 3-30 PerSeptive Biosystems 160
- 3.2.2 Loading Samples 161
- 3-32 PerSeptive Biosystems 162
- 3.2.3 Loading Samples 163
- (Thin Layer Application) 163
- PB100265 164
- PB100508 165
- PB100509 165
- PB100511 165
- PB100510 165
- 3.3 Cleaning Sample Plates 166
- Cleaning Sample Plates 167
- 3-38 PerSeptive Biosystems 168
- 3.4 Loading Sample Plates 169
- 3-40 PerSeptive Biosystems 170
- 3.4.2 Loading Sample Plates 172
- PB100278 173
- 65 66 173
- 74 75 76 173
- 84 85 86 173
- 3-44 PerSeptive Biosystems 174
- PB100277 175
- 3-46 PerSeptive Biosystems 176
- Sample Plate Types Supported 177
- 3-48 PerSeptive Biosystems 178
- 3-50 PerSeptive Biosystems 180
- .PLT files 181
- 3.5.3 Guidelines for 182
- Defining Custom Plate Types 182
- 3-54 PerSeptive Biosystems 184
- 3-56 PerSeptive Biosystems 186
- 3-58 PerSeptive Biosystems 188
- 3-60 PerSeptive Biosystems 190
- 3-62 PerSeptive Biosystems 192
- 3.5.4 Adjusting the Laser 193
- 3-64 PerSeptive Biosystems 194
- 4 Voyager 195
- Instrument Control 195
- Panel Basics 195
- 4.1 Instrument Control Panel 196
- Instrument Control Panel 197
- Workstation User’s Guide 4-3 197
- Control pages 198
- Voyager-DE STR system 198
- Spectrum window 199
- Status bar 199
- Figure 4-2 Status Bar 199
- Output window 199
- Displaying 200
- Manual control 200
- Workstation User’s Guide 4-7 201
- 4.2 Using the Control Pages 202
- Types of page 203
- 4-10 PerSeptive Biosystems 204
- Using the Spectrum Window 205
- 4-12 PerSeptive Biosystems 206
- 4.3.2 Zooming on Traces 207
- 4-14 PerSeptive Biosystems 208
- Figure 4-5 Adding Traces 209
- Removing traces 209
- 4.3.4 Annotating Traces 210
- 4-18 PerSeptive Biosystems 212
- 5. Click Print 213
- 4-20 PerSeptive Biosystems 214
- 4.4 Customizing the 215
- 4-22 PerSeptive Biosystems 216
- Instrument Menu Commands 218
- Controlling the Workstation 219
- 4-26 PerSeptive Biosystems 220
- Selecting Sample Position 221
- 4-28 PerSeptive Biosystems 222
- 4-30 PerSeptive Biosystems 224
- 4.6 Sequence Control Panel 226
- 4.7 How the Instrument 227
- Control Panels Interact 227
- 4-34 PerSeptive Biosystems 228
- Sequence Control Panel 229
- 4-36 PerSeptive Biosystems 230
- 5 Optimizing 231
- Instrument Settings 231
- 5.1 Loading, Modifying, and 232
- Saving Instrument Settings 232
- 5.1.2 Standard Instrument 233
- List of .BIC files 234
- Workstation User’s Guide 5-5 235
- 5-6 PerSeptive Biosystems 236
- 5.1.3 Opening and Viewing 237
- 5.1.4 Modifying an 238
- Modifying for 239
- Manual Control 239
- 5-10 PerSeptive Biosystems 240
- 5.1.5 Saving and Printing 241
- Saving .BIC files 242
- Printing 242
- Files to “Read-Only” Status 243
- 5.2 Instrument Settings 244
- Parameter Descriptions 244
- 5-16 PerSeptive Biosystems 246
- 5-18 PerSeptive Biosystems 248
- 5-20 PerSeptive Biosystems 250
- Data Explorer Software 251
- User’s Guide 251
- Matrix influence 252
- J. Am. Soc. Mass. Spectrom. 252
- Modifying the 253
- Instrument Mode Tab Displayed 254
- Voyager-DE system 256
- 5-28 PerSeptive Biosystems 258
- Advanced 259
- Advanced Tab Displayed 259
- 5-30 PerSeptive Biosystems 260
- 5-32 PerSeptive Biosystems 262
- 5-34 PerSeptive Biosystems 264
- 5-36 PerSeptive Biosystems 266
- 5-38 PerSeptive Biosystems 268
- 5.3.1 Summary of Parameters 269
- 5-40 PerSeptive Biosystems 270
- Potential gradient 272
- Maximum allowed 273
- Adjusting 273
- 5-44 PerSeptive Biosystems 274
- Relationship to 275
- Grid Voltage% 275
- Figure 5-10 Beam Guide Wire 276
- All models 276
- Linear mode 276
- Reflector mode 277
- PSD mode 277
- Horizontal 277
- 5-48 PerSeptive Biosystems 278
- Effects of 279
- Resolution and Data Quality 279
- • Input Bandwidth 280
- Suggested settings 281
- 5-52 PerSeptive Biosystems 282
- Bandwidth setting 283
- 5.4 Optimizing Instrument 284
- Settings Parameters 284
- 5.4.1 Optimization Strategy 285
- Strategy 286
- Laser intensity 287
- Adjusting laser 288
- General strategy 288
- Signal saturation 289
- 5-60 PerSeptive Biosystems 290
- 5.4.3 Optimizing Resolution 291
- 5.4.3.1 Overview 292
- 5-64 PerSeptive Biosystems 294
- Delayed Extraction Mode 295
- For more 296
- • Optimizing Delay Time 297
- 5-68 PerSeptive Biosystems 298
- Voltage% Values 299
- Time and Grid Voltage% Values 299
- 5-70 PerSeptive Biosystems 300
- 5-72 PerSeptive Biosystems 302
- 5-74 PerSeptive Biosystems 304
- 5.4.4.1 Overview 306
- 5-78 PerSeptive Biosystems 308
- 5-80 PerSeptive Biosystems 310
- 5-82 PerSeptive Biosystems 312
- 6 Acquiring 313
- Mass Spectra 313
- 6.1 Before You Begin 314
- Before You Begin 315
- Workstation User’s Guide 6-3 315
- 6-4 PerSeptive Biosystems 316
- Workstation User’s Guide 6-5 317
- 6-6 PerSeptive Biosystems 318
- Types of 319
- When to calibrate 320
- Workstation User’s Guide 6-9 321
- Data Explorer 322
- Software User’s Guide 322
- 6.2.1 Manually Acquiring 323
- 6-12 PerSeptive Biosystems 324
- 6-14 PerSeptive Biosystems 326
- 6-16 PerSeptive Biosystems 328
- 6-18 PerSeptive Biosystems 330
- 6.3 Obtaining Good Spectra 332
- Lower laser power producing: 333
- 6.3.2 Laser Intensity 335
- 6.3.3 Parameters Affecting 336
- 6.4 Making Accurate 337
- Mass Measurements 337
- 6-26 PerSeptive Biosystems 338
- 6.5.1 Detecting 339
- 6-28 PerSeptive Biosystems 340
- Height for most applications 341
- 6-30 PerSeptive Biosystems 342
- ____ ____ 343
- 6-32 PerSeptive Biosystems 344
- (± AMU) 345
- 5. Click OK 345
- 6.6.1 Before Acquiring in 346
- Automatic Control Mode 346
- Control mode 348
- Spectrum 350
- 6-40 PerSeptive Biosystems 352
- Continued 353
- Saving the 354
- 6.6.4 Search Pattern Files 356
- 0,0 1270,0-1270,0 357
- Default search 358
- 6-48 PerSeptive Biosystems 360
- Acquisition in Automatic Mode 361
- 6-50 PerSeptive Biosystems 362
- 6-52 PerSeptive Biosystems 364
- Control dialog box is used 365
- 6-54 PerSeptive Biosystems 366
- 6.6.6 Process that Occurs 368
- Accumulating All Spectra 369
- Accumulating Passing Spectra 370
- 6.7 Acquiring Spectra from 372
- Macros, and Calibration 374
- How file types 375
- 6-64 PerSeptive Biosystems 376
- 6-66 PerSeptive Biosystems 378
- 6.7.3 Creating a Sequence 379
- 6-68 PerSeptive Biosystems 380
- 6.7.3.2 Creating a Run List 381
- View menu 382
- 6-72 PerSeptive Biosystems 384
- 6-74 PerSeptive Biosystems 386
- 6-76 PerSeptive Biosystems 388
- 6.7.5 Running a Sequence 390
- 6-80 PerSeptive Biosystems 392
- Checking status 393
- Control Panel 393
- 6.7.7 Automatic Calibration 394
- During a Sequence Run 394
- Optimum Mass Accuracy 396
- 6-86 PerSeptive Biosystems 398
- 6.7.7.4 Internal Standard 399
- Calibration Considerations 399
- 6-88 PerSeptive Biosystems 400
- Using Separate Spots 401
- Using Workbook 402
- Showing or hiding 403
- Arranging 403
- 6-92 PerSeptive Biosystems 404
- 7 PSD Analysis 405
- 7.1 Overview of PSD Analysis 406
- Overview of PSD Analysis 407
- Workstation User’s Guide 7-3 407
- Reflector 408
- Immonium ions 409
- J. Am. Soc. Mass Spectrom. 409
- Chapter 7 PSD Analysis 410
- 7-6 PerSeptive Biosystems 410
- 7.1.3 PSD Data Files 411
- PSD calibration 412
- Default PSD 412
- Workstation User’s Guide 7-9 413
- Configuration Dialog Box 414
- 7.2 Enhancing 415
- Fragmentation with CID 415
- Benefits 416
- Gas requirements 417
- Meth. Enzymol 417
- 7-14 PerSeptive Biosystems 418
- 7-16 PerSeptive Biosystems 420
- 7.3 Acquiring PSD Data 421
- Settings (.BIC) Files 421
- Differences from 422
- Generating the 423
- 7-20 PerSeptive Biosystems 424
- 7.3.2 Determining the 425
- 7-22 PerSeptive Biosystems 426
- 7-24 PerSeptive Biosystems 428
- 7-26 PerSeptive Biosystems 430
- 7-28 PerSeptive Biosystems 432
- 7-30 PerSeptive Biosystems 434
- 7-32 PerSeptive Biosystems 436
- Changing 438
- 7.4 Exploring PSD Mode 440
- 7.4.1 Observing the 441
- Effects of Laser Intensity 441
- 7-38 PerSeptive Biosystems 442
- Exploring PSD Mode 443
- 7.4.2 Observing the Effects 444
- 7-42 PerSeptive Biosystems 446
- Figure 7-14 Ion Focusing 448
- 7-46 PerSeptive Biosystems 450
- 7.4.4 Summary 451
- 7.5 Viewing PSD Data 452
- 8 Maintenance and 453
- Troubleshooting 453
- 8.1.1 Maintenance Schedule 454
- 8.1.2 Hardware Maintenance 455
- Changing fuses 456
- Figure 8-1 Changing Fuses 457
- (5 x 20 mm) 458
- 8.2 Troubleshooting 459
- 8-8 PerSeptive Biosystems 460
- Workstation User’s Guide 8-9 461
- NOTE: Input Bandwidth is not 462
- NOTE: Data acquired using 463
- 8-12 PerSeptive Biosystems 464
- (continued on next page) 465
- (continued) 466
- NOTE: Increasing the laser 468
- 8-18 PerSeptive Biosystems 470
- 8-20 PerSeptive Biosystems 472
- Checking the 473
- Windows NT 473
- Event Log 473
- 8-22 PerSeptive Biosystems 474
- 8-24 PerSeptive Biosystems 476
- 8-26 PerSeptive Biosystems 478
- Appendix 479
- Appendix A Specifications 480
- Voyager-DE Specifications 481
- Workstation User’s Guide A-3 481
- Specifications 482
- Voyager-DE PRO Specifications 483
- Workstation User’s Guide A-5 483
- Voyager-DE STR Specifications 485
- Workstation User’s Guide A-7 485
- Workstation User’s Guide A-9 487
- B.1 Limited Product Warranty 490
- Workstation User’s Guide B-3 491
- B-4 PerSeptive Biosystems 492
- B.2 Damages, Claims, Returns 493
- B.3 Spare Parts 494
- Sample plates 495
- Membrane, Gels V700698 496
- Description Part Number 496
- Figure C-2 498
- (DHBs) Matrix Spectrum 499
- Appendix C Matrices 500
- C-4 PerSeptive Biosystems 500
- Workstation User’s Guide C-5 501
- if sample 502
- Workstation User’s Guide C-7 503
- PB100253 504
- Workstation User’s Guide C-9 505
- C-10 PerSeptive Biosystems 506
- C-12 PerSeptive Biosystems 508
- Sample Log 510
- Appendix D Log Sheets 516
- Elite, linear 518
- F.1 Calibration Compounds 520
- Calibration Compounds 521
- Workstation User’s Guide F-3 521
- F.2 Conversion of Mass to 522
- Time for Typical Standards 522
- Workstation User’s Guide F-5 523
- F.3 Theoretical 524
- Cleavages for Angiotensin 524
- F.4 Observed 525
- PSD Fragments in Angiotensin 525
- F-8 PerSeptive Biosystems 526
- Maintenance Log 528
- H.1 Optimizing a Continuous 530
- (.BIC) Setting 530
- Workstation User’s Guide H-3 531
- (.BIC) Files 532
- Optimization 533
- Optimizing 533
- Extraction Mode 534
- Continuous Extraction Mode 535
- Ratio, and Laser Threshold 536
- Workstation User’s Guide H-9 537
- H-10 PerSeptive Biosystems 538
- H.2.2.1 Overview 540
- H-14 PerSeptive Biosystems 542
- H-16 PerSeptive Biosystems 544
- H.2.3 Checking Resolution 545
- H-18 PerSeptive Biosystems 546
- H-20 PerSeptive Biosystems 548
- H-22 PerSeptive Biosystems 550
- Oscilloscope 554
- I.1 Guidelines for Acquiring 555
- I.2 Scaling 556
- Workstation User’s Guide I-5 557
- I-6 PerSeptive Biosystems 558
- I.3 Using the Control Stick 559
- I-8 PerSeptive Biosystems 560
- Bibliography 561
- Delayed Extraction 562
- MALDI Applications 562
- MS/MS Interpretation 563
- Peptide Applications 564
- DNA Applications 564
- Glossary 567
- Numerics 573
- Index-2 PerSeptive Biosystems 574
- Index-4 PerSeptive Biosystems 576
- Index-6 PerSeptive Biosystems 578
- Index-8 PerSeptive Biosystems 580
- Vertical Scale 5-28 583
- Vertical Scale 5-28, 5-50 594
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