Mass Spectrometry An Applied Approach

Provides a comprehensive description of mass spectrometry basics, applications, and perspectives

Mass spectrometry is a modern analytical technique, allowing for fast and ultrasensitive detection and identification of chemical species. It can serve for analysis of narcotics, counterfeit medicines, components of explosives, but also in clinical chemistry, forensic research and anti-doping analysis, for identification of clinically relevant molecules as biomarkers of various diseases. This book describes everything readers need to know about mass spectrometry—from the instrumentation to the theory and applications. It looks at all aspects of mass spectrometry, including inorganic, organic, forensic, and biological MS (paying special attention to various methodologies and data interpretation). It also contains a list of key terms for easier and faster understanding of the material by newcomers to the subject and test questions to assist lecturers. 

Knowing how crucial it is for young researchers to fully understand both the power of mass spectrometry and the importance of other complementary methodologies, Mass Spectrometry: An Applied Approach teaches that it should be used in conjunction with other techniques such as NMR, pharmacological tests, structural identification, molecular biology, in order to reveal the true function(s) of the identified molecule.

• Provides a description of mass spectrometry basics, applications and perspectives of the technique
• Oriented to a broad audience with limited or basic knowledge in mass spectrometry instrumentation, theory, and its applications in order to enhance their competence in this field
• Covers all aspects of mass spectrometry, including inorganic, organic, forensic, and biological MS with special attention to application of various methodologies and data interpretation
• Includes a list of key terms, and test questions, for easier and faster understanding of the material 

Mass Spectrometry: An Applied Approach is highly recommended for advanced students, young scientists, and anyone involved in a field that utilizes the technique.

Marek Smoluch, PhD, is an associate professor in the Department of Biochemistry and Neurobiology at AGH University of Science and Technology, Krakow, Poland.

Giuseppe Grasso, PhD, is an associate professor in the Department of Chemical Sciences at the University of Catania, Catania, Italy.

Piotr Suder, PhD, is an associate professor in the Department of Biochemistry and Neurobiology at AGH University of Science and Technology, Krakow, Poland.

Jerzy Silberring, PhD, is a professor and the Head of the Department of Biochemistry and Neurobiology at AGH University of Science and Technology, Krakow, Poland.

List of Contributors xvii

Preface xxi

1 Introduction 1
Jerzy Silberring and Marek Smoluch

2 A Brief History of Mass Spectrometry 5
Marek Smoluch and Jerzy Silberring

3 Basic Definitions 9
Marek Smoluch and Kinga Piechura

4 Instrumentation 13

4.1 Ionization Methods 13

4.1.1 Electron Ionization (EI) 13
Claudio Iacobucci

4.1.2 Chemical Ionization (CI) 15
Claudio Iacobucci

4.1.2.1 Principle of Operation: Positive and Negative Ion Modes 15

4.1.3 Atmospheric Pressure Ionization (API) 21

4.1.3.1 Atmospheric Pressure Chemical Ionization (APCI) 21
Claudio Iacobucci

4.1.3.2 Electrospray Ionization (ESI) 22
Piotr Suder

4.1.3.3 Nanoelectrospray 38
Piotr Suder

4.1.3.4 Desorption Electrospray Ionization (DESI) 42
Anna Bodzon‐Kulakowska and Anna Antolak

4.1.3.5 Laser Ablation Electrospray Ionization (LAESI) 49
Anna Bodzon‐Kulakowska and Anna Antolak

4.1.3.6 Photoionization 51
Jerzy Silberring

4.1.4 Ambient Plasma‐Based Ionization Techniques 54
Marek Smoluch

4.1.4.1 Introduction 54

4.1.4.2 Direct Analysis in Real Time (DART) 54

4.1.4.3 Flowing Atmospheric Pressure Afterglow (FAPA) 59

4.1.4.4 Dielectric Barrier Discharge Ionization (DBDI) 61

4.1.5 Matrix‐Assisted Laser Desorption/Ionization (MALDI) 64

4.1.5.1 Introduction 64
Przemyslaw Mielczarek and Jerzy Silberring

4.1.5.2 The Role of Matrix 66
Przemyslaw Mielczarek and Jerzy Silberring

4.1.5.3 Atmospheric Pressure MALDI 67
Giuseppe Grasso

4.1.5.4 MALDI Mass Spectra Interpretation 71
Przemyslaw Mielczarek and Jerzy Silberring

4.1.5.5 Desorption/Ionization on Porous Silicon (DIOS) 72
Przemyslaw Mielczarek and Jerzy Silberring

4.1.5.6 Surface‐Enhanced Laser Desorption/Ionization (SELDI) 73
Przemyslaw Mielczarek and Jerzy Silberring

4.1.5.7 Nanostructure‐Enhanced Laser Desorption/Ionization (NALDI) 74
Przemyslaw Mielczarek and Jerzy Silberring

4.1.5.8 Summary 75
Przemyslaw Mielczarek and Jerzy Silberring

4.1.6 Inductively Coupled Plasma Ionization (ICP) 78
Aleksandra Pawlaczyk and Małgorzata Iwona Szynkowska

4.1.6.1 Introduction 78

4.1.6.2 ICP as a Technique of Elemental Analysis and ICP Principle 78

4.1.6.3 Ionization of Elements and Ionization Efficiency 81

4.1.6.4 Mechanism of ICP Formation 82

4.1.6.5 Ways of Plasma View and Plasma Generation 84

4.1.6.6 Sample Introduction 84

4.1.6.7 Measurement in the ICP‐MS Technique 87

4.1.6.8 Analyzers in ICP‐MS Spectrometers 87

4.1.7 Secondary Ion Mass Spectrometry with Time‐of‐Flight Analyzer (TOF‐SIMS) 93
Nunzio Tuccitto

4.1.7.1 Introduction 93

4.1.7.2 TOF‐SIMS Principle of Operation 93

4.1.7.3 The Sputtering of the Sample Surface 94

4.1.7.4 Ionization (Generating Secondary Ions) 95

4.1.7.5 Construction of TOF‐SIMS 96

4.1.7.6 Analytical Capabilities of TOF‐SIMS 98

4.1.7.7 Examples and Spectra Interpretation 102

4.2 Analyzers 107

4.2.1 Time of Flight (TOF) 107
Anna Bodzon-Kulakowska and Anna Antolak

4.2.1.1 Introduction 107

4.2.1.2 The Working Rule of TOF Analyzer 108

4.2.1.3 Linear Mode of Operation of TOF 109

4.2.1.4 The Spread of the Kinetic Energy Regarding the Ions of the Same Mass 110

4.2.1.5 Delayed Ion Extraction 111

4.2.1.6 The Reflection Mode 113

4.2.1.7 Orthogonal Acceleration TOF Analyzer 114

4.2.1.8 Summary 116

4.2.2 Ion Mobility Analyzer (IM) 118
Anna Antolak and Anna Bodzon-Kulakowska

4.2.2.1 Principle of IM Operation 118

4.2.2.2 Drift Time IMS 118

4.2.2.3 High Field Asymmetric Waveform Ion Mobility Spectrometer (FAIMS) 119

4.2.2.4 Traveling Wave Ion Guides (TWIG) 121

4.2.2.5 IM Spectrum 122

4.2.2.6 Applications 122

4.2.3 Quadrupole Mass Analyzer 124
Anna Antolak and Anna Bodzon-Kulakowska 124

4.2.3.1 Construction and Principles of Operation of a Quadrupole 124

4.2.3.2 Behavior of an Ion Inside the Quadrupole 126

4.2.3.3 How Mass Spectrum Is Generated? Changes of U and V 128

4.2.3.4 Spectrum Quality 128

4.2.3.5 Applications of the Quadrupole Analyzer 129

4.2.3.6 Quadrupoles, Hexapoles, and Octapoles as Focusing Elements: Ion Guides 129

4.2.4 Ion Trap (IT) 131
Anna Bodzon-Kulakowska and Anna Antolak

4.2.4.1 Introduction 131

4.2.4.2 Behavior of an Ion Inside the Ion Trap 132

4.2.4.3 Analysis of the Ions 133

4.2.4.4 Mass Selective Instability Mode 134

4.2.4.5 Resonant Ejection Mode 135

4.2.4.6 Axial Modulation 137

4.2.4.7 Nonlinear Resonance 137

4.2.4.8 Linear Ion Trap (LIT) 137

4.2.4.9 Applications 139

4.2.5 High‐Resolution Mass Spectrometry 141
Piotr Stefanowicz and Zbigniew Szewczuk

4.2.5.1 Introduction 141

4.2.6 Ion Cyclotron Resonance (ICR) 142
Piotr Stefanowicz and Zbigniew Szewczuk

4.2.6.1 Introduction 142

4.2.6.2 Cyclotron Frequency 142

4.2.6.3 ICR: Principles of Operation 143

4.2.6.4 Injection of Ions into the ICR Cell 144

4.2.6.5 Trapping Electrodes 145

4.2.6.6 Excitation Electrodes 145

4.2.6.7 Detection Electrodes and Fourier Transform 145

4.2.6.8 FT‐ICR Properties as m/z Analyzer 147

4.2.7 Orbitrap 150
Piotr Stefanowicz and Zbigniew Szewczuk

4.2.7.1 History of Development and Principles of Operation 150

4.2.7.2 Analyzing Ions in the Orbitrap 151

4.2.7.3 Orbitrap Properties as m/z Analyzer 152

4.2.7.4 Analytical and Proteomic Applications of Orbitrap 153

4.2.8 Hybrid Mass Spectrometers 158
Giuseppe Di Natale

4.2.8.1 A Brief Comparison of Mass Analyzers 158

4.2.8.2 Triple Quadrupoles 159

4.2.8.3 Q‐IT 162

4.2.8.4 Q‐Orbitrap 162

4.2.8.5 Q‐TOF 163

4.2.8.6 IT‐TOF 165

4.2.8.7 IT‐Orbitrap 165

4.2.9 Sector Instruments 169
Anna Antolak and Anna Bodzon-Kulakowska

4.2.9.1 Introduction 169

4.2.9.2 Rule of Operation of Magnetic Analyzer (B) 169

4.2.9.3 Electrostatic Sector (E) 172

4.2.9.4 Mass Spectrometers with Magnetic and Electrostatic Sector 174

4.3 Ion Detectors 176

4.3.1 Introduction 176

4.3.2 Electron Multiplier 176

4.3.3 Microchannel Detector 177

4.3.4 Medipix/Timepix Detector 178

4.3.5 Ion Detection in ICR and Orbitrap‐Based Mass Spectrometers 179

5 Hyphenated Techniques 181

5.1 Gas Chromatography Combined with Mass Spectrometry (GC‐MS) 181
Anna Drabik

5.1.1 Introduction 181

5.1.2 Detectors 183

5.1.3 Chemical Modifications: Derivatization 186

5.1.4 GC‐MS Analysis 186

5.1.5 Two‐Dimensional Gas Chromatography Linked to Mass Spectrometry 2D GC‐MS 187

5.2 Liquid Chromatography Linked to Mass Spectrometry (LC‐MS) 193

5.2.1 Introduction 193
Francesco Bellia

5.2.2 Introduction to Liquid Chromatography 193
Anna Drabik

5.2.3 Types of Detectors 195
Anna Drabik

5.2.4 Chromatographic Columns 197
Anna Drabik

5.2.5 Chromatographic Separation and Quantitation Using MS as a Detector 200
Anna Drabik

5.2.6 Construction of an Interface Linking Liquid Chromatograph to the Mass Spectrometer 202
Anna Drabik 202

5.2.6.1 Introduction 202

5.2.6.2 ESI Interface 203

5.2.6.3 APCI Connection to MS 204

5.2.6.4 APPI Interface 205

5.2.6.5 LC Connection to MALDI‐MS 205

5.2.6.6 Multidimensional Separations 206

5.3 Capillary Electrophoresis Linked to Mass Spectrometry 209
Przemysław Mielczarek and Jerzy Silberring

5.3.1 Introduction 209

5.3.2 Types of Electrophoretic Techniques 210

5.3.3 Capillary Electrophoresis Linked to ESI 211

5.3.3.1 Introduction 211

5.3.3.2 Liquid Sheath Connection 212

5.3.3.3 Sheath‐Free Connection 212

5.3.3.4 Liquid Junction 213

5.3.4 Capillary Electrophoresis Linked to Matrix‐Assisted Laser Desorption/Ionization 214

5.3.4.1 Offline CE‐MALDI‐TOF 214

5.3.4.2 Direct CE‐MALDI‐TOF 214

5.3.4.3 Online CE‐MALDI‐TOF 215

5.3.5 Summary 215

6 Mass Spectrometry Imaging 217
Anna Bodzon‐Kulakowska and Anna Antolak

6.1 Introduction 217

6.2 SIMS 218

6.3 MALDI‐IMS 220

6.4 DESI 221

6.5 Analysis of Tissue Sections Using MSI Techniques 221

6.6 Analysis of Individual Cells and Cell Cultures Using MSI Techniques 223

6.7 Analysis with MSI Techniques: Examples 224

6.8 Combinations of Different Imaging Techniques 225

6.9 Summary 227

7 Tandem Mass Spectrometry 231
Piotr Suder

7.1 Introduction 231

7.2 Principles 231

7.3 Strategies for MS/MS Experiments 233

7.3.1 Tandem in Space 233

7.3.2 Tandem in Time 234

7.3.3 Multiple Fragmentation 236

7.4 Fragmentation Techniques 236

7.4.1 Introduction 236

7.4.2 (Low‐Energy) Collision‐Induced Dissociation (CID) 237

7.4.3 High‐Energy Collisional Dissociation (HCD) 237

7.4.4 Pulsed Q Collision‐Induced Dissociation (PQD) 238

7.4.5 Electron Capture Dissociation (ECD) 239

7.4.6 Electron Transfer Dissociation (ETD) 239

7.4.7 Electron Detachment Dissociation (EDD) 241

7.4.8 Negative Electron Transfer Dissociation (NETD) 241

7.4.9 Infrared Multiphoton Dissociation (IRMPD) 241

7.4.10 Blackbody Infrared Radiative Dissociation (BIRD) 242

7.4.11 Post‐source Decay (PSD): Metastable Ion Dissociation 242

7.4.12 Surface‐Induced Dissociation (SID) 243

7.4.13 Charge Remote Fragmentation 243

7.4.14 Chemically Activated Fragmentation (CAF) 243

7.4.15 Proton Transfer Reaction (PTR) 244

7.5 Practical Aspects of Fragmentation in Mass Spectrometers 245

7.5.1 In‐Source Fragmentation 245

7.5.2 Triple Quadrupole Fragmentation 246

7.5.3 Ion Traps 249

7.5.4 Time‐of‐Flight Analyzers 250

7.5.5 Combined Time‐of‐Flight Analyzers (TOF/TOF) 251

7.5.6 Hybrid Instruments 252

7.5.7 Mass Spectrometers Equipped with Orbitrap Analyzer 253

7.6 Applications of Tandem Mass Spectrometry in Life Sciences 254

7.7 SWATH Fragmentation 256

8 Mass Spectrometry Applications 261

8.1 Mass Spectrometry in Proteomics 261

8.1.1 Introduction 261
Vincenzo Cunsolo and Salvatore Foti

8.1.2 Bottom‐Up Versus Top‐Down Proteomics 262
Vincenzo Cunsolo and Salvatore Foti

8.1.2.1 Bottom‐Up Proteomics 262

8.1.2.2 Top‐Down Proteomics 265

8.1.3 Database Search and Protein Identification 267

8.1.4 In‐Depth Structural Characterization of a Single Protein: An Example 269

8.1.5 Quantitative Analysis in Proteomics 273
Joanna Ner‐Kluza, Anna Drabik, and Jerzy Silberring

8.1.5.1 Introduction 273

8.1.5.2 Isobaric Tags for Relative and Absolute Quantitation (iTRAQ) 274

8.1.5.3 Isotope‐Coded Affinity Tagging (ICAT) 276

8.1.5.4 Stable Isotope Labeling in Culture (SILAC) 279

8.1.5.5 Stable Isotope Labeling of Mammals (SILAM) 280

8.1.5.6 Mass‐Coded Abundance Tagging (MCAT) 281

8.1.5.7 Label‐Free Techniques 281

8.2 Food Proteomics 285
Vera Muccilli and Rosaria Saletti

8.3 Challenges in Analysis of Omics Data Generated by Mass Spectrometry 293
Katarzyna Pawlak, Emma Harwood, Fang Yu, and Pawel Ciborowski

8.3.1 Introduction 293

8.3.1.1 How Big Must Big Data Be? 294

8.3.1.2 Do Omics Experiments Generate Unstructured Data? 294

8.3.2 Targeted and Full Unbiased Omics Analysis Based on MS Technology 295

8.3.2.1 Factors Affecting Data Quality 296

8.3.2.2 Speed of MS Data Acquisition: Why Does It Matter? 297

8.3.2.3 Analytical Strategies in Omics Studies 298

8.3.3 Data Analysis and Visualization of Mass Spectrometry Omics Data 300

8.3.3.1 A Brief Introduction to Data Visualization 301

8.3.3.2 Exploration and Preparation of Data for Downstream Statistics and Visualization 304

8.3.3.3 Differential Expression Analysis 305

8.3.3.4 Strategies for Visualization Beyond Three Dimensions 310

8.3.3.5 Bioinformatics Tools 312

8.3.4 Databases and Search Algorithms 315

8.3.4.1 Databases for Proteomics 315

8.3.5 Validation of High‐Throughput Data: Current Challenges 318

8.3.5.1 Analytical Validation 319

8.3.5.2 Statistical Validation 320

8.3.5.3 Bioinformatics Validation 321

8.3.6 Summary and Conclusions 322

8.4 Application of the Mass Spectrometric Techniques in the Earth Sciences 326
Robert Anczkiewicz

8.4.1 Introduction 326

8.4.2 Conventional Geochronology 326

8.4.3 In Situ Geochronology 327

8.4.4 Geochemical and Isotopic Tracing 331

8.5 Mass Spectrometry in Space 335
Kathrin Altweg

8.5.1 Solar Wind and Plasma 339

8.5.2 Atmospheres of Planets and Moons 339

8.5.3 Comets 340

8.5.4 Interstellar and Cometary Dust 341

8.6 Mass Spectrometry in the Study of Art and Archaeological Objects 345
Giuseppe Spoto

8.6.1 Introduction 345

8.6.2 MS Methods for the Study of Inorganic Components of Art and Archaeological Objects 345

8.6.3 MS Methods for the Study of Organic Components of Art and Archaeological Objects 346

8.7 Application of ICP‐MS for Trace Elemental and Speciation Analysis 351
Aleksandra Pawlaczyk and Małgorzata Iwona Szynkowska

8.7.1 Introduction 351

8.7.2 Speciation Analysis by ICP‐MS: Examples of Applications 352

8.7.3 Single‐Particle and Single‐Cell Analysis by ICP‐MS: Examples of Applications 353

8.7.4 Imaging by LA‐ICP‐MS Technique 355

8.7.5 Improvements of LA‐ICP‐MS Technique 358

8.7.6 LA‐ICP Mass Spectrometer with LIBS 359

8.8 Mass Spectrometry in Forensic Research 362
Marek Smoluch and Jerzy Silberring

8.8.1 Introduction 362

8.8.2 Forgery in Art 362

8.8.3 Psychoactive Substances and Narcotics 363

8.8.4 Counterfeit Drugs and Generation of Metabolites 366

8.8.5 Terrorism/Explosives/Chemical Warfare 367

8.8.6 Future Prospects 368

8.9 Doping in Sport 372
Dorota Kwiatkowska

8.10 Miniaturization in Mass Spectrometry 384
Marek Smoluch and Jerzy Silberring

9 Appendix 389
Kinga Piechura and Marek Smoluch 389

9.1 Pressure Units 389

9.2 Most Commonly Detected Fragments Generated by Electron Impact (EI) Ionization 389

9.3 Trypsin Autolysis Products 393

9.4 Proteolytic Enzymes for Protein Identification 394

9.5 Molecular Masses of Amino Acid Residues 395

9.6 Molecular Masses of Less Common Amino Acid Residues 397

9.7 Internet Databases 400

9.7.1 Literature Databases 400

9.7.2 Scientific Journals 401

9.7.2.1 Journals Related to Mass Spectrometry 402

9.7.3 Bioinformatics Databases 402

9.7.3.1 Protein Databases 402

9.7.3.2 Database of Structures and Functions of Protein 403

9.7.3.3 Other Databases 404

9.7.4 Bioinformatics Tools 404

9.7.5 Useful Websites 405

10 Abbreviations 407
Kinga Piechura and Marek Smoluch

Index 413

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