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 xviiPreface xxi1 Introduction 1Jerzy Silberring and Marek Smoluch2 A Brief History of Mass Spectrometry 5Marek Smoluch and Jerzy Silberring3 Basic Definitions 9Marek Smoluch and Kinga Piechura4 Instrumentation 134.1 Ionization Methods 134.1.1 Electron Ionization (EI) 13Claudio Iacobucci4.1.2 Chemical Ionization (CI) 15Claudio Iacobucci4.1.2.1 Principle of Operation: Positive and Negative Ion Modes 154.1.3 Atmospheric Pressure Ionization (API) 214.1.3.1 Atmospheric Pressure Chemical Ionization (APCI) 21Claudio Iacobucci4.1.3.2 Electrospray Ionization (ESI) 22Piotr Suder4.1.3.3 Nanoelectrospray 38Piotr Suder4.1.3.4 Desorption Electrospray Ionization (DESI) 42Anna Bodzon‐Kulakowska and Anna Antolak4.1.3.5 Laser Ablation Electrospray Ionization (LAESI) 49Anna Bodzon‐Kulakowska and Anna Antolak4.1.3.6 Photoionization 51Jerzy Silberring4.1.4 Ambient Plasma‐Based Ionization Techniques 54Marek Smoluch4.1.4.1 Introduction 544.1.4.2 Direct Analysis in Real Time (DART) 544.1.4.3 Flowing Atmospheric Pressure Afterglow (FAPA) 594.1.4.4 Dielectric Barrier Discharge Ionization (DBDI) 614.1.5 Matrix‐Assisted Laser Desorption/Ionization (MALDI) 644.1.5.1 Introduction 64Przemyslaw Mielczarek and Jerzy Silberring4.1.5.2 The Role of Matrix 66Przemyslaw Mielczarek and Jerzy Silberring4.1.5.3 Atmospheric Pressure MALDI 67Giuseppe Grasso4.1.5.4 MALDI Mass Spectra Interpretation 71Przemyslaw Mielczarek and Jerzy Silberring4.1.5.5 Desorption/Ionization on Porous Silicon (DIOS) 72Przemyslaw Mielczarek and Jerzy Silberring4.1.5.6 Surface‐Enhanced Laser Desorption/Ionization (SELDI) 73Przemyslaw Mielczarek and Jerzy Silberring4.1.5.7 Nanostructure‐Enhanced Laser Desorption/Ionization (NALDI) 74Przemyslaw Mielczarek and Jerzy Silberring4.1.5.8 Summary 75Przemyslaw Mielczarek and Jerzy Silberring4.1.6 Inductively Coupled Plasma Ionization (ICP) 78Aleksandra Pawlaczyk and Małgorzata Iwona Szynkowska4.1.6.1 Introduction 784.1.6.2 ICP as a Technique of Elemental Analysis and ICP Principle 784.1.6.3 Ionization of Elements and Ionization Efficiency 814.1.6.4 Mechanism of ICP Formation 824.1.6.5 Ways of Plasma View and Plasma Generation 844.1.6.6 Sample Introduction 844.1.6.7 Measurement in the ICP‐MS Technique 874.1.6.8 Analyzers in ICP‐MS Spectrometers 874.1.7 Secondary Ion Mass Spectrometry with Time‐of‐Flight Analyzer (TOF‐SIMS) 93Nunzio Tuccitto4.1.7.1 Introduction 934.1.7.2 TOF‐SIMS Principle of Operation 934.1.7.3 The Sputtering of the Sample Surface 944.1.7.4 Ionization (Generating Secondary Ions) 954.1.7.5 Construction of TOF‐SIMS 964.1.7.6 Analytical Capabilities of TOF‐SIMS 984.1.7.7 Examples and Spectra Interpretation 1024.2 Analyzers 1074.2.1 Time of Flight (TOF) 107Anna Bodzon-Kulakowska and Anna Antolak4.2.1.1 Introduction 1074.2.1.2 The Working Rule of TOF Analyzer 1084.2.1.3 Linear Mode of Operation of TOF 1094.2.1.4 The Spread of the Kinetic Energy Regarding the Ions of the Same Mass 1104.2.1.5 Delayed Ion Extraction 1114.2.1.6 The Reflection Mode 1134.2.1.7 Orthogonal Acceleration TOF Analyzer 1144.2.1.8 Summary 1164.2.2 Ion Mobility Analyzer (IM) 118Anna Antolak and Anna Bodzon-Kulakowska4.2.2.1 Principle of IM Operation 1184.2.2.2 Drift Time IMS 1184.2.2.3 High Field Asymmetric Waveform Ion Mobility Spectrometer (FAIMS) 1194.2.2.4 Traveling Wave Ion Guides (TWIG) 1214.2.2.5 IM Spectrum 1224.2.2.6 Applications 1224.2.3 Quadrupole Mass Analyzer 124Anna Antolak and Anna Bodzon-Kulakowska 1244.2.3.1 Construction and Principles of Operation of a Quadrupole 1244.2.3.2 Behavior of an Ion Inside the Quadrupole 1264.2.3.3 How Mass Spectrum Is Generated? Changes of U and V 1284.2.3.4 Spectrum Quality 1284.2.3.5 Applications of the Quadrupole Analyzer 1294.2.3.6 Quadrupoles, Hexapoles, and Octapoles as Focusing Elements: Ion Guides 1294.2.4 Ion Trap (IT) 131Anna Bodzon-Kulakowska and Anna Antolak4.2.4.1 Introduction 1314.2.4.2 Behavior of an Ion Inside the Ion Trap 1324.2.4.3 Analysis of the Ions 1334.2.4.4 Mass Selective Instability Mode 1344.2.4.5 Resonant Ejection Mode 1354.2.4.6 Axial Modulation 1374.2.4.7 Nonlinear Resonance 1374.2.4.8 Linear Ion Trap (LIT) 1374.2.4.9 Applications 1394.2.5 High‐Resolution Mass Spectrometry 141Piotr Stefanowicz and Zbigniew Szewczuk4.2.5.1 Introduction 1414.2.6 Ion Cyclotron Resonance (ICR) 142Piotr Stefanowicz and Zbigniew Szewczuk4.2.6.1 Introduction 1424.2.6.2 Cyclotron Frequency 1424.2.6.3 ICR: Principles of Operation 1434.2.6.4 Injection of Ions into the ICR Cell 1444.2.6.5 Trapping Electrodes 1454.2.6.6 Excitation Electrodes 1454.2.6.7 Detection Electrodes and Fourier Transform 1454.2.6.8 FT‐ICR Properties as m/z Analyzer 1474.2.7 Orbitrap 150Piotr Stefanowicz and Zbigniew Szewczuk4.2.7.1 History of Development and Principles of Operation 1504.2.7.2 Analyzing Ions in the Orbitrap 1514.2.7.3 Orbitrap Properties as m/z Analyzer 1524.2.7.4 Analytical and Proteomic Applications of Orbitrap 1534.2.8 Hybrid Mass Spectrometers 158Giuseppe Di Natale4.2.8.1 A Brief Comparison of Mass Analyzers 1584.2.8.2 Triple Quadrupoles 1594.2.8.3 Q‐IT 1624.2.8.4 Q‐Orbitrap 1624.2.8.5 Q‐TOF 1634.2.8.6 IT‐TOF 1654.2.8.7 IT‐Orbitrap 1654.2.9 Sector Instruments 169Anna Antolak and Anna Bodzon-Kulakowska4.2.9.1 Introduction 1694.2.9.2 Rule of Operation of Magnetic Analyzer (B) 1694.2.9.3 Electrostatic Sector (E) 1724.2.9.4 Mass Spectrometers with Magnetic and Electrostatic Sector 1744.3 Ion Detectors 1764.3.1 Introduction 1764.3.2 Electron Multiplier 1764.3.3 Microchannel Detector 1774.3.4 Medipix/Timepix Detector 1784.3.5 Ion Detection in ICR and Orbitrap‐Based Mass Spectrometers 1795 Hyphenated Techniques 1815.1 Gas Chromatography Combined with Mass Spectrometry (GC‐MS) 181Anna Drabik5.1.1 Introduction 1815.1.2 Detectors 1835.1.3 Chemical Modifications: Derivatization 1865.1.4 GC‐MS Analysis 1865.1.5 Two‐Dimensional Gas Chromatography Linked to Mass Spectrometry 2D GC‐MS 1875.2 Liquid Chromatography Linked to Mass Spectrometry (LC‐MS) 1935.2.1 Introduction 193Francesco Bellia5.2.2 Introduction to Liquid Chromatography 193Anna Drabik5.2.3 Types of Detectors 195Anna Drabik5.2.4 Chromatographic Columns 197Anna Drabik5.2.5 Chromatographic Separation and Quantitation Using MS as a Detector 200Anna Drabik5.2.6 Construction of an Interface Linking Liquid Chromatograph to the Mass Spectrometer 202Anna Drabik 2025.2.6.1 Introduction 2025.2.6.2 ESI Interface 2035.2.6.3 APCI Connection to MS 2045.2.6.4 APPI Interface 2055.2.6.5 LC Connection to MALDI‐MS 2055.2.6.6 Multidimensional Separations 2065.3 Capillary Electrophoresis Linked to Mass Spectrometry 209Przemysław Mielczarek and Jerzy Silberring5.3.1 Introduction 2095.3.2 Types of Electrophoretic Techniques 2105.3.3 Capillary Electrophoresis Linked to ESI 2115.3.3.1 Introduction 2115.3.3.2 Liquid Sheath Connection 2125.3.3.3 Sheath‐Free Connection 2125.3.3.4 Liquid Junction 2135.3.4 Capillary Electrophoresis Linked to Matrix‐Assisted Laser Desorption/Ionization 2145.3.4.1 Offline CE‐MALDI‐TOF 2145.3.4.2 Direct CE‐MALDI‐TOF 2145.3.4.3 Online CE‐MALDI‐TOF 2155.3.5 Summary 2156 Mass Spectrometry Imaging 217Anna Bodzon‐Kulakowska and Anna Antolak6.1 Introduction 2176.2 SIMS 2186.3 MALDI‐IMS 2206.4 DESI 2216.5 Analysis of Tissue Sections Using MSI Techniques 2216.6 Analysis of Individual Cells and Cell Cultures Using MSI Techniques 2236.7 Analysis with MSI Techniques: Examples 2246.8 Combinations of Different Imaging Techniques 2256.9 Summary 2277 Tandem Mass Spectrometry 231Piotr Suder7.1 Introduction 2317.2 Principles 2317.3 Strategies for MS/MS Experiments 2337.3.1 Tandem in Space 2337.3.2 Tandem in Time 2347.3.3 Multiple Fragmentation 2367.4 Fragmentation Techniques 2367.4.1 Introduction 2367.4.2 (Low‐Energy) Collision‐Induced Dissociation (CID) 2377.4.3 High‐Energy Collisional Dissociation (HCD) 2377.4.4 Pulsed Q Collision‐Induced Dissociation (PQD) 2387.4.5 Electron Capture Dissociation (ECD) 2397.4.6 Electron Transfer Dissociation (ETD) 2397.4.7 Electron Detachment Dissociation (EDD) 2417.4.8 Negative Electron Transfer Dissociation (NETD) 2417.4.9 Infrared Multiphoton Dissociation (IRMPD) 2417.4.10 Blackbody Infrared Radiative Dissociation (BIRD) 2427.4.11 Post‐source Decay (PSD): Metastable Ion Dissociation 2427.4.12 Surface‐Induced Dissociation (SID) 2437.4.13 Charge Remote Fragmentation 2437.4.14 Chemically Activated Fragmentation (CAF) 2437.4.15 Proton Transfer Reaction (PTR) 2447.5 Practical Aspects of Fragmentation in Mass Spectrometers 2457.5.1 In‐Source Fragmentation 2457.5.2 Triple Quadrupole Fragmentation 2467.5.3 Ion Traps 2497.5.4 Time‐of‐Flight Analyzers 2507.5.5 Combined Time‐of‐Flight Analyzers (TOF/TOF) 2517.5.6 Hybrid Instruments 2527.5.7 Mass Spectrometers Equipped with Orbitrap Analyzer 2537.6 Applications of Tandem Mass Spectrometry in Life Sciences 2547.7 SWATH Fragmentation 2568 Mass Spectrometry Applications 2618.1 Mass Spectrometry in Proteomics 2618.1.1 Introduction 261Vincenzo Cunsolo and Salvatore Foti8.1.2 Bottom‐Up Versus Top‐Down Proteomics 262Vincenzo Cunsolo and Salvatore Foti8.1.2.1 Bottom‐Up Proteomics 2628.1.2.2 Top‐Down Proteomics 2658.1.3 Database Search and Protein Identification 2678.1.4 In‐Depth Structural Characterization of a Single Protein: An Example 2698.1.5 Quantitative Analysis in Proteomics 273Joanna Ner‐Kluza, Anna Drabik, and Jerzy Silberring8.1.5.1 Introduction 2738.1.5.2 Isobaric Tags for Relative and Absolute Quantitation (iTRAQ) 2748.1.5.3 Isotope‐Coded Affinity Tagging (ICAT) 2768.1.5.4 Stable Isotope Labeling in Culture (SILAC) 2798.1.5.5 Stable Isotope Labeling of Mammals (SILAM) 2808.1.5.6 Mass‐Coded Abundance Tagging (MCAT) 2818.1.5.7 Label‐Free Techniques 2818.2 Food Proteomics 285Vera Muccilli and Rosaria Saletti8.3 Challenges in Analysis of Omics Data Generated by Mass Spectrometry 293Katarzyna Pawlak, Emma Harwood, Fang Yu, and Pawel Ciborowski8.3.1 Introduction 2938.3.1.1 How Big Must Big Data Be? 2948.3.1.2 Do Omics Experiments Generate Unstructured Data? 2948.3.2 Targeted and Full Unbiased Omics Analysis Based on MS Technology 2958.3.2.1 Factors Affecting Data Quality 2968.3.2.2 Speed of MS Data Acquisition: Why Does It Matter? 2978.3.2.3 Analytical Strategies in Omics Studies 2988.3.3 Data Analysis and Visualization of Mass Spectrometry Omics Data 3008.3.3.1 A Brief Introduction to Data Visualization 3018.3.3.2 Exploration and Preparation of Data for Downstream Statistics and Visualization 3048.3.3.3 Differential Expression Analysis 3058.3.3.4 Strategies for Visualization Beyond Three Dimensions 3108.3.3.5 Bioinformatics Tools 3128.3.4 Databases and Search Algorithms 3158.3.4.1 Databases for Proteomics 3158.3.5 Validation of High‐Throughput Data: Current Challenges 3188.3.5.1 Analytical Validation 3198.3.5.2 Statistical Validation 3208.3.5.3 Bioinformatics Validation 3218.3.6 Summary and Conclusions 3228.4 Application of the Mass Spectrometric Techniques in the Earth Sciences 326Robert Anczkiewicz8.4.1 Introduction 3268.4.2 Conventional Geochronology 3268.4.3 In Situ Geochronology 3278.4.4 Geochemical and Isotopic Tracing 3318.5 Mass Spectrometry in Space 335Kathrin Altweg8.5.1 Solar Wind and Plasma 3398.5.2 Atmospheres of Planets and Moons 3398.5.3 Comets 3408.5.4 Interstellar and Cometary Dust 3418.6 Mass Spectrometry in the Study of Art and Archaeological Objects 345Giuseppe Spoto8.6.1 Introduction 3458.6.2 MS Methods for the Study of Inorganic Components of Art and Archaeological Objects 3458.6.3 MS Methods for the Study of Organic Components of Art and Archaeological Objects 3468.7 Application of ICP‐MS for Trace Elemental and Speciation Analysis 351Aleksandra Pawlaczyk and Małgorzata Iwona Szynkowska8.7.1 Introduction 3518.7.2 Speciation Analysis by ICP‐MS: Examples of Applications 3528.7.3 Single‐Particle and Single‐Cell Analysis by ICP‐MS: Examples of Applications 3538.7.4 Imaging by LA‐ICP‐MS Technique 3558.7.5 Improvements of LA‐ICP‐MS Technique 3588.7.6 LA‐ICP Mass Spectrometer with LIBS 3598.8 Mass Spectrometry in Forensic Research 362Marek Smoluch and Jerzy Silberring8.8.1 Introduction 3628.8.2 Forgery in Art 3628.8.3 Psychoactive Substances and Narcotics 3638.8.4 Counterfeit Drugs and Generation of Metabolites 3668.8.5 Terrorism/Explosives/Chemical Warfare 3678.8.6 Future Prospects 3688.9 Doping in Sport 372Dorota Kwiatkowska8.10 Miniaturization in Mass Spectrometry 384Marek Smoluch and Jerzy Silberring9 Appendix 389Kinga Piechura and Marek Smoluch 3899.1 Pressure Units 3899.2 Most Commonly Detected Fragments Generated by Electron Impact (EI) Ionization 3899.3 Trypsin Autolysis Products 3939.4 Proteolytic Enzymes for Protein Identification 3949.5 Molecular Masses of Amino Acid Residues 3959.6 Molecular Masses of Less Common Amino Acid Residues 3979.7 Internet Databases 4009.7.1 Literature Databases 4009.7.2 Scientific Journals 4019.7.2.1 Journals Related to Mass Spectrometry 4029.7.3 Bioinformatics Databases 4029.7.3.1 Protein Databases 4029.7.3.2 Database of Structures and Functions of Protein 4039.7.3.3 Other Databases 4049.7.4 Bioinformatics Tools 4049.7.5 Useful Websites 40510 Abbreviations 407Kinga Piechura and Marek SmoluchIndex 413