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Chapter 3 Preparing Samples
3-10 PerSeptive Biosystems
3
THAP
Use THAP for small oligonucleotides <3,500 Da, acidic
carbohydrates, acidic glycopeptides, acid sensitive
compounds. THAP provides a more even response than
3-HPA.
Matrix concentration • Oligonucleotides—10 mg/ml
• Acidic carbohydrates—2 mg/ml
Additive concentration 50 mg/ml diammonium citrate in deionized water
Final sample concentration 1–10 pmol/µl
Solvents 50 percent acetonitrile and deionized water
NOTE: HPLC-grade water may vary in salt
concentration. Do not use for oligonucleotide
analysis.
Preparation
1
Follow the procedure in “Preparing matrix” on
page 3-5 and:
Small oligonucleotides:
Combine 8:1 THAP:diammonium citrate. Air dry
sample plate after loading sample and matrix.
Acidic carbohydrates and glycopeptides:
Combine 20:1 THAP:diammonium citrate. Dry
sample plate under vacuum after loading
sample and matrix. Wait a few minutes after
drying until the sample absorbs ambient
humidity and turns faint white.
Crystals Larger than sinapinic, overlapping shingles,
non-uniform shape (see Figure 3-2 on page 3-35)
Stability Prepare weekly.
1. Other special sample preparation techniques are described in Papac, D.I., A. Wong, A.J.S. Jones,
Anal. Chem.,
1996, 68, 3215–3223.
- 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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