List of Contributors ......................................... xvii
Preface ....................................................... xxi
Part I. Motivation and Essentials ............................... 1
Chapter 1 Introduction ......................................... 3
A kos Wertes and Károly Vékey
Chapter 2 Basics of Analytical Chemistry and Mass
Spectrometry for Medical Professionals .......................... 7
Károly Vékey and András Telekes
1 Introduction ................................................. 7
2 Terms and Definitions ........................................ 9
3 The Analytical Procedure .................................... 11
4 A Case Study: Analysis of Plasma Sterol Profile ............. 14
5 Mass Spectrometry ........................................... 16
Reference ...................................................... 18
Chapter 3 Ethical, Legal, Safety, and Scientific Aspects of
Medical Research ............................................... 19
András Telekes and Károly Vékey
1 Ethical Aspects ............................................. 20
2 Legal Aspects ............................................... 22
3 Safety Aspects .............................................. 22
4 Handling Biological Materials ............................... 25
5 Clinical Trials and Protocols ............................... 28
6 Administrative Procedures ................................... 33
References .................................................. 34
Part II. Tools of the Trade .................................... 35
Chapter 4 Biomedical Sampling ................................. 37
György Vas, Kornél Nagy, and Károly Vékey
1 Sampling .................................................... 37
2 Sample Preparation .......................................... 40
2.1 Centrifugation ......................................... 41
2.2 Filtration ............................................. 42
2.3 Protein Precipitation .................................. 42
2.4 Ultrafiltration ........................................ 42
2.5 Dialysis and Electrodialysis ........................... 43
2.6 Digestion .............................................. 44
2.7 Chemical Derivatization ................................ 45
2.8 Lyophilization ......................................... 45
3 Extraction Techniques ....................................... 46
3.1 Liquid-Liquid Extraction (LLE) ......................... 46
3.2 Solid-Phase Extraction (SPE) ........................... 48
3.3 ZipTip® Sampling ....................................... 53
3.4 Solid-Phase Microextraction ............................ 53
4 Automation and High Throughput .............................. 57
5 Outlook ..................................................... 58
References .................................................. 59
Chapter 5 Separation Methods .................................. 61
Kornél Nagy and Károly Vékey
1 Introduction ................................................ 61
2 Gas Chromatography .......................................... 72
3 High-Performance Liquid Chromatography ...................... 77
3.1 Normal-Phase Liquid Chromatography ..................... 80
3.2 Reverse-Phase Liquid Chromatography .................... 81
3.3 Ion-Exchange Liquid Chromatography ..................... 83
3.4 Size Exclusion Chromatography (Gel Filtration) ......... 84
3.5 2D Liquid Chromatography ............................... 84
4 Electrophoretic Techniques .................................. 85
4.1 Capillary Zone Electrophoresis ......................... 85
4.2 Capillary Gel Electrophoresis .......................... 86
4.3 Capillary Isoelectric Focusing ......................... 86
4.4 Sodium Dodecyl Sulfate Polyacrylamide Gel
Electrophoresis ........................................ 87
4.5 2D Gel Electrophoresis ................................. 87
5 Future Trends ............................................... 88
References .................................................. 89
Chapter 6 Mass Spectrometry Instrumentation and Techniques .... 93
Árpád Somogyi
1 Introduction ................................................ 94
2 General Questions about Mass Measurement and Mass
Spectrometry ................................................ 96
3 Separation Techniques: Gas Chromatography (GC), and High-
Performance Liquid Chromatography (HPLC) ................... 100
4 Ionization Methods ......................................... 102
4.1 Electron Impact (EI) Ionization ....................... 104
4.2 Chemical Ionization (CI) .............................. 108
4.3 Fast-Atom Bombardment (FAB) and Liquid Secondary Ion
Mass Spectrometry (LSIMS) ............................. 110
4.4 Electrospray Ionization (ESI) ......................... 111
4.5 Atmospheric Pressure Chemical Ionization (APCI) ....... 115
4.6 Matrix-Assisted Laser Desorption/Ionization (MALDI) ... 116
5 Mass Analyzers ............................................. 119
5.1 Time-of-Flight (TOF) Analyzers ........................ 120
5.2 Interaction with Electrostatic Fields: Electrostatic
(ESA) and Orbitrap (ОТ) Analyzers ..................... 123
5.3 Interactions with a Magnetic Field: Magnetic (B)
Analyzer .............................................. 124
5.4 Interactions with a Magnetic and Electrostatic
Field: Ion Cyclotron Resonance (ICR) Analyzers ........ 125
5.5 Interaction with Electromagnetic Fields: Quadrupole
(Q) Analyzers ......................................... 127
5.6 Interaction with Electromagnetic Fields: Linear
Ion-Trap Quadrupole (LTQ) Analyzers ................... 128
5.7 Interaction with Electromagnetic Fields: Three-
Dimensional Quadrupole Ion Trap (3D QIT) Analyzers .... 129
6 Tandem Mass Spectrometry (MS/MS) ........................... 130
7 Selected Terms for Clarification ........................... 135
Acknowledgment ............................................. 137
References ................................................. 137
Chapter 7 Chemoinformatics—Multivariate Mathematical-
Statistical Methods for Data Evaluation ....................... 141
Károly Héberger
1 Introduction ............................................... 141
2 Data Types and Data Pretreatment ........................... 143
2.1 Data Types ............................................ 143
2.2 Arrangement of Data ................................... 144
2.3 Data Pretreatment ..................................... 145
3 Multivariate Methods ....................................... 146
3.1. Principal Component Analysis (PCA) .................... 148
3.2 Cluster Analysis (CA) ................................. 149
3.3 Multiple Linear Regression (MLR) ...................... 151
3.4 Linear Discriminant Analysis (LDA) and Canonical
Correlation Analysis (CCA) ............................ 152
3.5 Partial Least Squares Projection of Latent
Structures (PLS) ...................................... 154
3.6 Classification and Regression Trees (CART) ............ 156
3.7 Artificial Neural Networks (ANN) ...................... 157
3.8 Some Methods of Variable Selection .................... 158
4 Selected Applications of Chemometrics ...................... 162
Terms and Terminology ...................................... 164
References ................................................. 166
Part III. Biomolecules ........................................ 171
Chapter 8 Mass Spectrometry in Proteomics .................... 173
Akos Wertes
1 Introduction ............................................... 173
2 Methods in Proteomics ...................................... 176
2.1 Peptide Mapping ....................................... 177
2.2 Peptide Fragmentation ................................. 179
2.3 Sequence Tags ......................................... 182
2.4 De Novo Sequencing .................................... 183
2.5 Electron Capture and Electron Transfer Dissociations .. 184
2.6 Quantitative Proteomics ............................... 186
2.7 Higher Order Structures ............................... 187
2.8 Mapping Protein Function .............................. 189
3 Outlook .................................................... 190
References ................................................. 191
Chapter 9 De Novo Sequencing of Peptides ..................... 195
Matthew T. Olson, Jonathan A. Epstein, and Alfred L. Yergey
Chapter 10 Protein Bioinformatics ............................. 203
Peter McGarvey, Hongzhan Huang, and Cathy H. Wu
1 Highlights for Medical Professionals ....................... 203
2 Introduction ............................................... 205
3 Methodology ................................................ 206
3.1 UniProt Sequence Databases ............................ 206
3.2 PIRSF Protein Family Classification ................... 207
3.3 iProClass Integrated Protein Database ................. 210
3.4 NIAID Proteomic Bioinformatics Resource ............... 212
4 Discussion ................................................. 214
5 Future Trends .............................................. 215
5.1 iProLINK Literature Mining Resource ................... 215
5.2 iProXpress Knowledge System for Gene Expression
and Proteomic Data Analysis ........................... 217
6 Conclusions ................................................ 219
Acknowledgments ............................................ 220
References ................................................. 220
Chapter 11 Analysis of Complex Lipidomes ...................... 223
Andreas Uphoff, Martin Hermansson, Perttu Haimi, and Pentti
Somerharju
1 Introduction ............................................... 224
2 Methodology ................................................ 226
2.1 Lipid Extraction ...................................... 226
2.2 Mass Spectrometry ..................................... 226
2.3 Data Analysis ......................................... 229
3 Phospholipids .............................................. 230
3.1 Phosphatidylcholine and Sphingomyelin ................. 230
3.2 Phosphatidylethanolamine .............................. 232
3.3 Phosphatidylserine .................................... 232
3.4 Phosphatidylglycerol, Lysobisphosphatidic Acid, and
Phosphatidic Acid ..................................... 233
3.5 Cardiolipin ........................................... 233
3.6 Phosphatidylinositol and Polyphosphoinositides ........ 234
4 Acylglycerols .............................................. 234
4.1 Triacylglycerols ...................................... 234
4.2 Diacylglycerols and Monoacylglycerols ................. 235
5 Sphingolipids .............................................. 235
5.1 Free Sphingoid Bases .................................. 235
5.2 Ceramides ............................................. 236
5.3 Neutral Glycosphingolipids ............................ 236
5.4 Sulfatides ............................................ 236
5.5 Gangliosides .......................................... 237
6 Sterols .................................................... 237
6.1 Cholesterol and Other Sterols ......................... 237
6.2 Steryl Esters and Steroid Hormones .................... 238
7 Medical Applications of MS-Lipidomics ...................... 238
7.1 Diagnostics and Therapy ............................... 238
7.2 Disease Mechanisms .................................... 239
7.3 Nutrition and Other Issues ............................ 239
8 Future Trends .............................................. 240
9 Conclusions ................................................ 240
References ................................................. 240
Part IV. Selected Medical Applications ........................ 251
Chapter 12 Medical Laboratory Practice—Possibilities for
Mass Spectrometry ............................................. 253
Olaf Bodamer
1 Introduction ............................................... 253
2 Quality Management ......................................... 254
3 Inborn Errors of Metabolism ................................ 254
3.1 Analysis of Homocysteine .............................. 255
3.2 Analysis of Organic Acids Including Orotic Acid ....... 257
3.3 Analysis of Oligosaccharides .......................... 257
3.4 Analysis of Lysosomal Enzyme Activities ............... 258
4 Assessment of In Vivo Metabolism Using Stable Isotope
Techniques ................................................. 258
5 Conclusions ................................................ 259
References ................................................. 259
Chapter 13 Therapeutic Drug Monitoring and Measurement
of Drug Concentrations Using Mass Spectrometry ................ 263
András Telekes, Márta Hegedűs, and István Kiss
1 Introduction ............................................... 263
2 Antiinfection Drugs ........................................ 267
3 Drugs Acting on the Central Nervous System (CNS) ........... 271
4 Cardiovascular Drags ....................................... 273
5 Anticancer Agents .......................................... 276
6 Analgesics ................................................. 280
7 Miscellaneous Drug Classes ................................. 283
References ................................................. 285
Chapter 14 Mass Spectrometry of Infectious Pathogens .......... 291
Plamen A. Demirev
1 Introduction ............................................... 291
1.1 Highlights for Medical Professionals .................. 293
1.2 Highlights for Chemists ............................... 294
2 Methodology ................................................ 295
2.1 MALDI MS-Based Methods for Bacillus Spore Species
Characterization ...................................... 295
2.2 LD MS Detection of Plasmodium Parasites in Blood ...... 301
2.3 Other MS-Based Methods for Pathogen Detection/
Identification ........................................ 304
3 Future Trends and Prospects ................................ 305
References ................................................. 305
Chapter 15 Proteomics of Viruses .............................. 309
Anne Pumfery, Reem Berro, and Fatah Kashanchi
1 Introduction ............................................... 310
1.1 Highlights for Medical Professionals .................. 310
1.2 Highlights for Chemists ............................... 314
2 Virus-Host Interactions .................................... 317
2.1 Proteomics of Herpesvirus Virions ..................... 318
2.2 Proteomics of Epstein-Barr Virus ...................... 319
2.3 Proteomics of Herpes Simplex Virus .................... 321
2.4 Proteomics of Retroviruses—HIV and HTLV ............... 322
2.5 Proteomics of Hepatitis С Virus and Hepatocellular
Carcinoma ............................................. 324
3 Diagnostics ................................................ 325
3.1 2DE-MS: SARS, HBV, HCV, and HIV-1 ..................... 325
3.2 LC-MS: HIV-1 and HCV .................................. 327
3.3 SELDI ProteinChip: SARS, HIV, and Hepatitis ........... 328
3.4 Protein Microarray: Vaccinia Virus .................... 330
4 Discussion ................................................. 331
5 Future Trends .............................................. 334
6 Conclusions ................................................ 335
Acknowledgments ............................................ 336
References ................................................. 336
Chapter 16 Neonatal Research .................................. 345
Bruno Casetta
1 Introduction ............................................... 346
1.1 Essential Medical Concepts ............................ 346
1.2 Basic Concept of Using LC-MS/MS Technology in the
Clinical Domain ....................................... 347
2 Principle of the Methodology ............................... 348
3 Quick Reference for Practical Implementation of the
Methodology ................................................ 349
3.1 Extraction ............................................ 350
3.2 Butylation ............................................ 350
3.3 Analytical Equipment .................................. 351
3.4 Analytical Measurement ................................ 351
3.5 Expected Performances ................................. 352
4 Key Points of the Methodology .............................. 356
4.1 Quantitation Assessment ............................... 356
4.2 Derivatization ........................................ 356
4.3 FIA Row Rate Regime ................................... 358
4.4 MS Scanning Strategies ................................ 362
4.5 Data Processing ....................................... 364
5 Ongoing Extensions of NBS .................................. 366
5.1 Extended Panel of Amino Acids ......................... 366
5.2 Very Long Chain Fatty Acids ........................... 369
5.3 Steroids .............................................. 369
5.4 Bile Acids ............................................ 371
6 Conclusions ................................................ 374
Acknowledgments ............................................ 376
References ................................................. 376
Chapter 17 Applications of Mass Spectrometry in Oncology ...... 379
John Roboz
1 Introduction ............................................... 380
2 New Methodology—SELDI-TOF-MS ............................... 381
2.1 Protein Chips ......................................... 381
2.2 Identification ........................................ 382
2.3 Proteomic Pattern Diagnostics ......................... 383
2.4 Problems and Prospects ................................ 384
3 Other Relevant Methodological Challenges ................... 384
3.1 Analysis of Cells ..................................... 384
3.2 Direct Tissue Analysis and Imaging MS ................. 384
3.3 The Problem of Dynamic Ranges ......................... 384
3.4 Low-Abundance, Low-Molecular Mass Proteins or Drugs
in Plasma/Serum ....................................... 385
3.5 Quantification ........................................ 385
4 Diagnostic Oncoproteomics Based on SELDI-TOF-MS ............ 386
4.1 Ovarian and Endometrial Cancer ........................ 387
4.2 Breast Cancer ......................................... 387
4.3 Prostate Cancer ....................................... 389
4.4 Pancreatic Cancer ..................................... 390
4.5 Bladder Cancer ........................................ 390
4.6 Head and Neck Cancer .................................. 391
4.7 Miscellaneous Malignancies ............................ 391
4.8 Other Searches for Biomarkers Using SELDI-TOF-MS ...... 393
5 Representative Other Applications .......................... 393
5.1 Proteomic Studies to Uncover Molecular Mechanisms
Associated with Malignancies .......................... 393
5.2 Proteomic Profiles to Provide Predictors of Drag-
Modulated Targets and Responses ....................... 394
5.3 Profiles to Identify Proteins Associated with
Disease Progression ................................... 394
5.4 Targeted Biomarker Detection Via Whole Protein
Analysis .............................................. 395
5.5 Sphingolipids in Cancer Pathogenesis and Treatment .... 395
5.6 Quantification of Antineoplastic Drags ................ 395
5.7 Helicobacter Pylori ................................... 395
5.8 Molecular Epidemiology for Chemoprevention ............ 397
5.9 Selenium .............................................. 397
References ................................................. 398
Chapter 18 Application of Mass Spectrometry in Brain
Research: Neurotransmitters, Neuropeptides, and Brain
Proteins ...................................................... 407
Laszlo Prokai
1 Introduction ............................................... 407
2 Methodology ................................................ 408
2.1 Neurotransmitters ..................................... 408
2.2 Neuropeptides ......................................... 409
2.3 Brain Proteins (Neuroproteomics) ...................... 411
3 Discussion ................................................. 417
3.1 Neurotransmitters ..................................... 417
3.2 Neuropeptides ......................................... 417
3.3 Neuroproteomics ....................................... 419
4 Future Trends .............................................. 420
5 Conclusions ................................................ 421
Acknowledgments ............................................ 421
References ................................................. 421
Chapter 19 The Human Pituitary Proteome: Clinical
Applications .................................................. 425
Xianquan Zhan, Harold Sacks, and Dominic M. Desiderio
1 Introduction ............................................... 426
1.1 Proteomics, Functional Proteomics, and Comparative
Proteomics ............................................ 426
1.2 The Pathophysiological Basis of Pituitary Adenoma
Comparative Proteomics ................................ 428
1.3 Basic Techniques Used for Studying Proteomics ......... 428
2 The Pituitary Gland and Mass Spectrometry: An
Endocrinologist's Perspective .............................. 430
3 Methodology ................................................ 432
3.1 Gel-Based Comparative Proteomics of Human Pituitary
Adenoma Tissues ....................................... 433
3.2 The Proteomics of PTM Proteins in Human Pituitary
Adenomas .............................................. 441
3.3 Challenge of Comparative Proteomics in the Study of
Human Pituitary Adenomas .............................. 445
4 Discussion ................................................. 446
4.1 Insights into the Basic Molecular Mechanisms of
Pituitary Tumor Formation ............................. 446
4.2 Discovery of Potential Biomarkers Related to
Pituitary Adenomas .................................... 449
4.3 Pituitary Hormone Isoforms in Human Pituitary
Adenomas .............................................. 449
5 Future Trends .............................................. 452
5.1 Combination of Gel-Proteomics and Non-Gel
Quantitative Proteomics ............................... 452
5.2 Comparative Proteomics Studies of PTM Proteins in
Human Pituitary Adenomas .............................. 452
5.3 Integration of Proteomics and Transcriptomics to
Study Human Pituitary Adenomas ........................ 453
5.4 Protein Chips Coupled with Mass Spectrometry to
Study Human Pituitary Adenomas ........................ 453
6 Conclusions ................................................ 454
Acknowledgments ............................................ 455
References ................................................. 455
Chapter 20 Mass Spectrometry of Proteinous Allergens
Inducing Human Diseases ....................................... 459
Martina Marchetti, Jasmin Hirschmann, Elisabeth
Förster-Waldl, and Günter Allmaier
1 Introduction ............................................... 459
1.1 Highlights for Medical Professionals .................. 462
1.2 Highlights for Chemists ............................... 463
2 Methodology ................................................ 465
3 Mass Spectrometric Identification of Proteinous Allergens .. 469
4 Future Trends .............................................. 477
5 Conclusions ................................................ 479
References ................................................. 480
Chapter 21 Mass Spectrometry in Clinical Treatment ............ 487
András Telekes, Márta Hegedűs, and István Kiss
1 Introduction ............................................... 487
2 Pediatrics ................................................. 490
3 Oncology ................................................... 493
References ................................................. 499
Part V. Emerging Areas ........................................ 503
Chapter 22 Biomarker Discovery ................................ 505
Bradley J. Thatcher and Emilia Caputo
1 Introduction ............................................... 506
1.1 Chemical Background for Medical Doctors ............... 506
1.2 Basic Medical Aspects ................................. 507
1.3 Basic Concepts ........................................ 507
2 Biomarkers in Medicine ..................................... 508
3 Important Definitions ...................................... 509
4 Biomarkers Discovery and Complexity of Biological Systems .. 510
5 Biomarker Discovery and "Omics" ............................ 512
6 The Biomarker Discovery Project ............................ 512
6.1 The Biomarker Discovery Group ......................... 512
6.2 The Biomarker Discovery Experiment .................... 513
7 Challenges in the Biomarker Discovery Pathway .............. 516
7.1 Asking the Right Question ............................. 516
7.2 Sample Availability ................................... 518
7.3 Which Sample Should Be Used? .......................... 519
7.4 Sample Management ..................................... 520
8 Technical Issues in Experimental Design .................... 522
8.1 Sample Preparation .................................... 522
8.2 Separation Technique .................................. 524
8.3 Bioinformatics ........................................ 524
8.4 Methods ............................................... 525
9 The discovery Process Overview ............................. 526
9.1 Tools for Biomarker Discovery ......................... 526
9.2 Biomarker Identification and Validation ............... 527
10 Conclusions ................................................ 530
References ................................................. 531
Chapter 23 Molecular Imaging by Mass Spectrometry ............. 533
Sarah A. Schwartz and Richard M. Caprioli
1 Introduction ............................................... 533
2 Methods .................................................... 535
2.1 Sample Preparation .................................... 535
2.2 Biological Applications ............................... 540
3 Discussion ................................................. 547
4 Future Trends .............................................. 549
5 Conclusion ................................................. 550
Acknowledgments ............................................... 551
References .................................................... 551
Chapter 24 Brief Outlook ...................................... 555
Károly Vékey, András Telekes, and Akos Vertes
1 Trends in Instrumentation .................................. 556
2 Emerging Systems Approach .................................. 558
3 Mass Spectrometry in Translational Medicine ................ 559
Index ......................................................... 561
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