Introduction to Mass Spectrometry. Instrumentation, Applications and Strategies for Data Interpretation. Fourth Edition

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7 INTRODUCTION TO MASS SPECTROMETRY Instrumentation, Applications and Strategies for Data Interpretation FOURTH EDITION J. THROCK WATSON Professor of Biochemistry and of Chemistry Michigan State University East Lansing, Michigan O. DAVID SPARKMAN Adjunct Professor of Chemistry College of the Pacific University of the Pacific Stockton, California John Wiley & Sons, Ltd

8 Copyright 2007 John Wiley & Sons Ltd, The Atrium, Southern Gate, Chichester, West Sussex PO19 8SQ, England Telephone (+44) (for orders and customer service enquiries): Visit our Home Page on or Reprinted with corrections March 2008 All Rights Reserved. No part of this publication may be reproduced, stored in a retrieval system or transmitted in any form or by any means, electronic, mechanical, photocopying, recording, scanning or otherwise, except under the terms of the Copyright, Designs and Patents Act 1988 or under the terms of a licence issued by the Copyright Licensing Agency Ltd, 90 Tottenham Court Road, London W1T 4LP, UK, without the permission in writing of the Publisher. Requests to the Publisher should be addressed to the Permissions Department, John Wiley & Sons Ltd, The Atrium, Southern Gate, Chichester, West Sussex PO19 8SQ, England, or ed to permreq@wiley.co.uk, or faxed to (+44) Designations used by companies to distinguish their products are often claimed as trademarks. All brand names and product names used in this book are trade names, service marks, trademarks or registered trademarks of their respective owners. The Publisher is not associated with any product or vendor mentioned in this book. This publication is designed to provide accurate and authoritative information in regard to the subject matter covered. It is sold on the understanding that the Publisher is not engaged in rendering professional services. If professional advice or other expert assistance is required, the services of a competent professional should be sought. The Publisher and the Author make no representations or warranties with respect to the accuracy or completeness of the contents of this work and specifically disclaim all warranties, including without limitation any implied warranties of fitness for a particular purpose. The advice and strategies contained herein may not be suitable for every situation. In view of ongoing research, equipment modifications, changes in governmental regulations, and the constant flow of information relating to the use of experimental reagents, equipment, and devices, the reader is urged to review and evaluate the information provided in the package insert or instructions for each chemical, piece of equipment, reagent, or device for, among other things, any changes in the instructions or indication of usage and for added warnings and precautions. The fact that an organization or Website is referred to in this work as a citation and/or a potential source of further information does not mean that the author or the publisher endorses the information the organization or Website may provide or recommendations it may make. Further, readers should be aware that Internet Websites listed in this work may have changed or disappeared between when this work was written and when it is read. No warranty may be created or extended by any promotional statements for this work. Neither the Publisher nor the Author shall be liable for any damages arising herefrom. Other Wiley Editorial Offices John Wiley & Sons Inc., 111 River Street, Hoboken, NJ 07030, USA Jossey-Bass, 989 Market Street, San Francisco, CA , USA Wiley-VCH Verlag GmbH, Boschstr. 12, D Weinheim, Germany John Wiley & Sons Australia Ltd, 42 McDougall Street, Milton, Queensland 4064, Australia John Wiley & Sons (Asia) Pte Ltd, 2 Clementi Loop #02-01, Jin Xing Distripark, Singapore John Wiley & Sons Ltd, 6045 Freemont Blvd, Mississauga, Ontario L5R 4J3, Canada Wiley also publishes its books in a variety of electronic formats. Some content that appears in print may not be available in electronic books. Anniversary Logo Design: Richard J. Pacifico Library of Congress Cataloging-in-Publication Data Watson, J. Throck. Introduction to mass spectrometry : instrumentation, applications, and strategies for data interpretation / J. Throck Watson, O. David Sparkman. -- 4th ed. p. cm. Includes index. ISBN (cloth) 1. Mass spectrometry. 2. Biomolecules--Analysis. I. Sparkman, O. David (Orrin David), II. Title. QC454.M3W '.65--dc British Library Cataloguing in Publication Data A catalogue record for this book is available from the British Library ISBN (H/B) Typeset by the authors Printed and bound in Great Britain by Antony Rowe, Chippenham, Wiltshire vi

9 Contents Preface... xix Acknowledgments... xxiii Chapter 1 Introduction... 1 I. Introduction The Tools and Data of Mass Spectrometry The Concept of Mass Spectrometry...4 II. History...9 III. Some Important Terminology Used In Mass Spectrometry Introduction Ions Peaks Resolution and Resolving Power...25 IV. Applications Example 1-1: Interpretation of Fragmentation Patterns (Mass Spectra) to Distinguish Positional Isomers Example 1-2: Drug Overdose: Use of GC/MS to Identify a Drug Metabolite Example 1-3: Verification that the Proper Derivative of the Compound of Interest Has Been Prepared Example 1-4: Use of a CI Mass Spectrum to Complement an EI Mass Spectrum Example 1-5: Use of Exact Mass Measurements to Identify Analytes According to Elemental Composition Example 1-6: Is This Protein Phosphorylated? If So, Where? Example 1-7: Clinical Diagnostic Tests Based on Quantitation of Stable Isotopes by Mass Spectrometry in Lieu of Radioactivity...42 V. The Need for Chromatography...42 VI. Closing Remarks...44 VII. Monographs on Mass Spectrometry Published Before Chapter 2 The Mass Spectrometer I. Introduction...55 II. Ion Guides...56 III. Types of m/z Analyzers Time-of-Flight m/z Analyzers...62 A. Linear ) Resolving Power of the Linear TOF Instrument ) Time-Lag Focusing ) Beam Deflection...67 B. Reflectron...69 C. Orthogonal Acceleration...74 vii

10 viii Contents D. Ion Detection in the TOF Analyzer ) Time-Slice Detection ) Time-Array Detection ) TAD with Transient Recorders ) TAD with an Integrating Transient Recorder ) Hadamard Transform TOF MS Quadrupole Ion Traps...82 A. 3D Quadrupole Ion Trap...84 B. Linear Quadrupole Ion Trap (LIT)...97 C. Performance Trade-Offs in the Ion Trap The Orbitrap A. Historical Aspects B. Operating Principles ) Role of the C Trap in Success of the Orbitrap ) Figures of Merit for the Orbitrap as an m/z Analyzer Transmission Quadrupoles A. QMF Equations of Motion B. The Stability Diagram C. Characteristics of Output D. Spectral Skewing E. Performance Limitations Magnetic-Sector Instruments A. Single-Focusing Instruments ) Operating Principles ) Magnetic Versus Scanning ) Performance Limitations B. Double-Focusing Instruments FTICR-MS A. Hardware Configuration B. Operational Considerations C. Representative Applications Ion Mobility Spectrometry (IMS) A. Operating Principles B. FAIMS C. Applications IV. Calibration of the m/z Scale Electron Ionization Chemical Ionization Electrospray Ionization and APCI Techniques MALDI V. Ion Detectors General Considerations Types of Detectors A. Faraday Cup B. Electron Multiplier ) Discrete-Dynode Version ) Continuous-Dynode Version C. Negative-Ion Detection D. Post-Acceleration Detection and Detection of High-Mass Ions E. Channel Electron Multiplier Array (CEMA)...144

11 Contents F. Electro-Optical Ion Detection G. The Daly Detector H. Cryogenic Detectors I. Ion Detection in FTMS VI. Vacuum Systems Introduction Definitions Pressure Gauges A. Thermal-Conductivity Gauges B. Pirani Gauge C. Thermocouple Gauges Ionization Gauges A. Hot-Cathode Gauge B. Cold-Cathode Gauge Types of Pumps A. Mechanical Pumps (Low Vacuum) ) Rotary Vane Pumps ) Scroll Pumps ) Roots Pumps ) Diaphram Pumps B. High Vacuum ) Turbomolecular Pumps ) Oil Diffusion Pumps ) Sputter-Ion Pumps (Nonregeneratable Getter Pumps) Chapter 3 Mass Spectrometry/Mass Spectrometry I. Introduction History and the Evolution of the Technique Concept and Definitions Nomenclature II. Ion Dissociation Metastable Ions Collisionally Activated Dissociation Electron Capture Dissociation Electron-Transfer Dissociation Illustrative Example of Qualitative Analysis by MS/MS III. Instrumentation for MS/MS Tandem-in-Space Mass Spectrometry (MS/MS) A. Triple-Quadrupole Mass Spectrometer B. Q-TOF Hybrid Mass Spectrometer C. TOF-TOF Mass Spectrometer D. BEqQ Hybrid Mass Spectrometer E. Double-Focusing Instrument Tandem-in-Time Mass Spectrometry IV. Specialized Techniques and Applications In-Source CAD CAD in Conjunction with Soft Ionization A. Data-Dependent Acquisition ix

12 Contents 3. Selected Reaction Monitoring A. Illustrative Example Showing that SRM Has a Higher Specificity than SIM in Spite of a Lower Signal Strength B. An Example Comparing the Specificity of SRM and SIM in the Context of Analyzing a Biological Sample for a Drug Metabolite Precursor-Ion Analysis Neutral-Loss (Common Neutral-Loss) Analysis Ion/Molecule Reactions Hybrid Instrumentation for MS/MS and Ion Mobility Spectrometry (IMS) V. Analyte Identification from MS/MS Data Introduction Identifying an Unknown Using a Product-Ion Mass Spectrum Similarities between EI and Product-Ion Mass Spectra Another Way of Using Substructure Identification Searching of Product-Ion Spectra against Standardized Databases VI. Concluding Remarks about MS/MS Chapter 4 Inlet Systems I. Introduction II. Batch Inlets Heated Reservoir Inlet Direct Inlet Probe (DIP) A. The Chromatoprobe Direct Exposure Probe (Desorption Chemical Ionization, DCI) Pyrolysis III. Continuous Inlets Membrane Introduction MS (MIMS) Supercritical Fluid Chromatography (SFC) Electrophoretic Inlet IV. Ionization Inlet Systems Direct Analysis in Real Time (DART) Desorption Electrospray Ionization (DESI) Desorption Atmospheric Pressure Chemical Ionization (DAPCI) V. Speciality Interfaces Selected Ion Flow Tube Mass Spectrometry (SIFTMS) Fast Atom Bombardment (FAB) and Liquid Secondary Ion Mass Spectrometry (LSIMS) Chemical Reaction Interface Mass Spectrometry (CRIMS) Inductively Coupled Plasma Mass Spectrometry (ICPMS) A. Hardware Configuration B. Operational Considerations C. Electrothermal Vaporization D. Laser Ablation E. Speciation F. Summary VI. Final Statement x

13 Contents Chapter 5 Strategies for Data Interpretation (Other than Fragmentation) I. Introduction II. Some Important Definitions III. Possible Information That Can Be Obtained from the Mass Spectrum IV. Elemental Composition of an Ion and the Ratios of Its Isotope Peaks Definition of Terms Related to the Matter of Mass Spectrometry Nitrogen Rule Elemental Composition of an Ion Based on the Ratio of Isotope Peak Intensities A. Isotope Peak Patterns Used to Determine the Elemental Composition of Ions B. Isotope Peak Patterns for Ions Containing Various Combinations of Br/Cl C. Constraint on the Number of Atoms Allowed for a Given Element D. Relationship of the Charge State of an Ion and the Spacing of the Corresponding Isotope Peaks ) Ions of High Mass-to-Charge Ratio E. Steps to Assigning an Elemental Composition Based on Isotope Peak Intensities F. Validating the Putative Elemental Composition of an Ion G. An Illustrative Example of the Use of Isotope Peak Ratios to Determine an Elemental Composition H. Potential Problems Arising from Adjacent Peaks Elemental Composition as a Function of an Accurate Determination of the m/z Value of a Mass Spectral Peak A. Appearance of Mass Spectra of High-m/z Value Ions Using EI Data to Identify Unknowns Detected During Analysis by LC/MS Does the Result Make Sense? V. Identifying the Mass of an Analyte Recognition of the Peak Representing the Molecular Ion in EI A. Reasonable Losses from the Molecular Ion in EI Recognition of the Protonated Molecule (MH ) in Soft Ionization A. Probable Adducts Observed in the Mass Spectrum Produced by Soft Ionization Recognition of the Deprotonated Molecule ([M H] ) Peak in Soft Ionization VI. Recognition of Spurious Peaks in the Mass Spectrum Noise Spikes Peaks Corresponding to Contaminants in GC/MS and LC/MS A. The Phthalate Ion Peak B. GC Column Bleed C. Cluster Ions VII. Obtaining Structural Information from the Mass Spectrum xi

14 xii Contents Chapter 6 Electron Ionization I. Introduction II. Ionization Process III. Strategy for Data Interpretation Assumptions The Ionization Process IV. Types of Fragmentation Pathways Sigma-Bond Cleavage Homolytic or Radical-Site-Driven Cleavage Heterolytic or Charge-Site-Driven Cleavage Rearrangements A. Hydrogen-Shift Rearrangements B. Hydride-Shift Rearrangements V. Representative Fragmentations (Spectra) of Classes of Compounds Hydrocarbons A. Saturated Hydrocarbons ) Straight-Chain Hydrocarbons ) Branched Hydrocarbons ) Cyclic Hydrocarbons B. Unsaturated C. Aromatic Alkyl Halides Oxygen-Containing Compounds A. Aliphatic Alcohols B. Aliphatic Ethers C. Aromatic Alcohols D. Cyclic Ethers E. Ketones and Aldehydes F. Aliphatic Acids and Esters G. Aromatic Acids and Esters Nitrogen-Containing Compounds A. Aliphatic Amines B. Aromatic Compounds Containing Atoms of Nitrogen C. Heterocyclic Nitrogen-Containing Compounds D. Nitro Compounds E. Concluding Remarks on the Mass Spectra of Nitrogen-Containing Compounds Multiple Heteroatoms or Heteroatoms and a Double Bond Trimethylsilyl Derivative Determining the Location of Double Bonds VI. Library Searches and EI Mass Spectral Databases Databases Library Search Programs What To Do When the Spectrum of the Unknown is Not in the Database(s) Searching Multiple Databases Database Size and Quality Concluding Remarks on the NIST Mass Spectral Search Program VII. Summary of Interpretation of EI Mass Spectra...442

15 Contents Chapter 7 Chemical Ionization I. Introduction II. Description of the Chemical Ionization Source III. Production of Reagent Ions from Various Reagent Gases IV. Positive-Ion Formation Under CI Fundamentals Practical Consideration of Proton Affinity in CI Selective Ionization Fragmentation V. Negative-Ion Formation under CI True Negative Chemical Ionization Resonant Electron Capture Negative Ionization VI. Data Interpretation and Systematic Studies of CI VII. Ionization by Charge Exchange Mechanism of Ionization Fragmentation and Appearance of Mass Spectra VIII. Atmospheric Pressure Chemical Ionization IX. Desorption Chemical Ionization X. General Applications XI. Concluding Remarks Chapter 8 Electrospray Ionization I. Introduction II. Operating Principles III. Appearance of ESI Mass Spectra and Data Interpretation IV. ESI with an m/z Analyzer of High Resolving Power V. Conventional ESI Source Interface VI. Nanoelectrospray and Microelectrospray Ionization VII. Desorption Electrospray Ionization (DESI) VIII. Effect of Composition and Flow Rate of an Analyte Solution IX. Special Applications Direct Analysis of Ions in Solution by ESI Cold-Spray Ionization Negative-Ion Detection Secondary Electrospray Ionization (SESI) Kinetic Measurements of Chemical Reactions ESI Generation of Ions for Ancillary Experiments X. General Applications of ESI Chapter 9 MALDI I. Historical Perspective and Introduction II. Operating Principles The Matrix The Laser, m/z Analyzer, and Representative Mass Spectra The Ionization Process High-Pressure (HP) MALDI and Atmospheric Pressure (AP) MALDI xiii

16 Contents III. Sample Handling Sample Preparation of the Conventional Plate The Problem of Analyte Solubility The Problem of Sample Purity On-Probe Sample Purification and/or Modification A. SAMs and Polymer-Modified Surfaces B. Affinity Surfaces Direct Analysis from Gels Hydrogen/Deuterium Exchange IV. Special Instrumental Techniques Post-Source Decay (PSD) Ion Excitation Delayed Extraction (DE) Desorption Ionization On Silicon (DIOS) Tissue Profiling or Imaging V. Representative Applications Proteins and Peptides Microbes Biomarkers Synthetic Polymers Small Molecules Quantitation Combined with Liquid Chromatography Chapter 10 Gas Chromatography/Mass Spectrometry I. Introduction II. Introduction to GC Basic Types of Injectors Injection Considerations and Syringe Handling Syringeless Modes of Sample Injection for Fast GC III. Sample Handling Proper Sample Container Analyte Isolation and Purification Derivative Formation A. Silyl Derivatives B. Esters of Carboxylic Acids C. Oxime Derivatives D. Acyl Derivatives E. Derivatives for Characterizing Double Bonds IV. Instrument Requirements for GC/MS Operating Pressures Typical Parameters for a Conventional GC-MS Interface Supersonic Molecular Beam Interface for GC/MS Open-Split Interface Molecular Separators A. Jet-Orifice Separator B. Membrane Separator Inertness of Materials in the Interface xiv

17 Contents V. Operational Considerations Spectral Skewing Background/Bleed The Need for Rapid Acquisition of Mass Spectra A. Performance Trade-Offs of Conventional Instruments for GC/MS B. Time-Array Detection Selected Ion Monitoring (SIM) A. Definition and Nomenclature B. Development of the Technique C. Qualitative Example of SIM D. Quantitative Example of SIM E. Mechanics of Ion Monitoring ) Adjustment of the Mass Scale ) Mass Range ) Magnetic Mass Spectrometer ) Transmission Quadrupole Mass Spectrometer ) Number of Ion Currents (Masses) F. Programmable SIM G. SIM at High Resolving Power VI. Sources of Error Errors Relating to Equipment or Procedure Errors Relating to Contamination Sources of Interference Dealing with Background in a Mass Spectrum A. AMDIS (Automated Mass spectral Deconvolution and Identification System) B. Other Software Techniques VII. Representative Applications of GC/MS VIII. Special Techniques Purge and Trap Thermal Desorption Chapter 11 Liquid Chromatography/Mass Spectrometry I. Introduction II. Historical Milestones in the Development of the Interface Introduction The Direct Inlet The Moving-Belt Interface The Thermospray Interface Continuous-Flow FAB III. Currently Viable Versions of the Interface Atmospheric Pressure Ionization A. Electrospray Ionization Interface ) Optimization for Analyses by HPLC ) Capillary Electrophoresis Interface B. APCI Interface xv

18 Contents C. APPI Interface ) Operating Principles of APPI ) Operating Mechanics for APPI ) Signal Suppression ) Applications of APPI Particle Beam Interface Electron Ionization and LC/MS IV. Special Operation of LC under MS Conditions Effects of Mobile-Phase Composition A. Signal Suppression B. Use of Internal Standards in the Face of Signal Suppression C. Adjusting the Chromatography in the Face of Signal Suppression during LC/MS D. Ion Pairing and Signal Suppression E. Influence of the Type and the Nature of LC Buffer F. Influence of Solvent Composition on the ESI Signal G. Adduct Formation H. Spectral Interference I. System Compromise Differences in Method Development for ESI vs APCI V. Applications Attention to High Throughput Chapter 12 Analysis of Proteins and Other Biopolymers I. Introduction II. Proteins Sequencing A. Nomenclature and Fragmentation in Sequencing of Peptides ) Nomenclature ) Fragmentation B. Strategy for Deducing Amino Acid Sequence via CAD of Peptides ) An Illustrative Example ) Possible Pitfalls in Interpretation ) Search for Confirming Ions ) Ladder Sequencing Mass Mapping A. Peptide Mass Fingerprinting B. De novo Sequencing C. Sequence Tagging D. Sequest E. Evaluation of Hits in Automated Searches F. Data-Dependent Analysis by Mass Spectrometry Post-Translational Modifications A. Recognition of Sites of Protein Phosphorylation ) An Illustrative Example ) Selective Capture and Detection of Phosphopeptides ) Chemical Modification of Phosphorylation Sites xvi

19 Contents B. Recognition of Sites of Sulfation C. Recognition of Sites of Glycosylation D. Acetylation of Lysine E. Cysteine Status in Proteins ) Are There Any Disulfide Bonds? ) Which Cysteines Are Free? ) What Is the Linkage of Cysteines in the Disulfide Bonds? (A) Conventional Proteolytic Mass Mapping of Disulfides (B) Cyanylation-Based Mass Mapping of Disulfides F. Recognition of Ubiquinated Proteins G. Other Types of Modifications Quantitation in Proteomics A. ICATs ) Operating Principles ) Illustrative Example of the ICAT Approach ) Analogous to ICAT Methodologies B. Alternative Stable Isotope-Based Methodologies C. Related Methodologies Top-Down Strategies of Analysis A. Instrumentation and Fragmentation Requirements B. Electron Capture Dissociation (ECD) C. Electron-Transfer Dissociation (ETD) D. Applications Noncovalent Interactions Folding and Unfolding Applications III. Oligonucleotides Analytical Considerations Sequencing A. Nomenclature B. Algorithm for Data Interpretation Applications IV. Carbohydrates Analytical Considerations Nomenclature Diagnostic Fragmentation Applications Subject Index xvii

20 xviii Contents

21 PREFACE This edition of Introduction to Mass Spectrometry is far more than a revision of the third edition, which appeared in Completely updated and more than 75% rewritten, it covers strategies for data interpretation, fundamental operating principles of instrumentation, and representative applications for all areas of organic, environmental, and biomedical mass spectrometry. A majority of the chapters have bibliographies containing several hundred references to research articles and reviews, mostly published since Most chapters, but especially the first two, provide a historical perspective on the development of mass spectrometry as well as commentary on the evolution of commercial developments of the instrumentation. Careful attention to nomenclature is provided throughout the book. In addition to serving as a general reference for the subject of mass spectrometry as it pertains to organic and biochemistry, this book is designed for use as a textbook for courses on mass spectrometry. The readily comprehensible approach to the topic, honed through the teamwork of the coauthors in teaching hundreds of classes on various aspects of mass spectrometry for nearly 30 years under the auspices of the American Chemical Society, will benefit the reader. The physical instrument is dissected and described in Chapter 2 in a systematic manner from the ion source through ion guides to the m/z analyzer to the detection system with attention to the vacuum system. The fundamental physics for each type of m/z analyzer, as well as for common detectors and vacuum pumps, are provided together with a common sense description of the operating principles of each. Chapter 3 describes the concept of MS/MS with emphasis on collisionally activated dissociation. Tandem-in-space is distinguished from tandem-in-time, and several qualitative and quantitative applications of both types of technology are presented in the context of environmental and biomedical fields. In addition, information on analyte identification from MS/MS is provided along with explanations and sources of spectral databases and how to use them. Various means of transporting the sample into the low-pressure environment of the mass spectrometer are described in Chapter 4. The operating mechanics of batch inlet systems as well as continuous sampling systems are presented together with representative and/or illustrative examples. Descriptions of nonchromatographic continuous inlets include DART, DESI, DAPCI, SIFT, MIMS, CRIMS, pyrolysis, electrophoresis, laser ablation, continuous-flow FAB, and ICP. Continuous inlets in combination with chromatography include SFC and pyrolysis GC and are presented in Chapters 10 and 11, respectively. A general strategy for interpretation of a mass spectrum, regardless of the type of ionization involved, is presented in Chapter 5. The Nitrogen Rule is introduced and used in a variety of situations. The importance of isotope peak-intensity ratios is introduced; several carefully detailed examples are described that show the relationship between isotope peak-intensity ratios and the elemental composition of the corresponding ion. The basis for recognizing peaks representing odd-electron vs even-electron ions is introduced; the importance of recognizing such ions is illustrated with appropriate examples of mass spectra resulting from a variety of ionization types, including EI, CI, and electrospray. xix

22 Preface Chapter 6 is one of the highlights of the book, providing a solid introduction to the formation, appearance, and interpretation of EI mass spectra. Emphasis is placed on recognizing the most probable site of electron deficiency (site of the, the plus/dot ) in the molecular ion, which is the precursor of a majority of the ions represented by the fragmentation pattern in an EI mass spectrum. Four major pathways of fragmentation of a molecular ion (sigma-bond cleavage, homolytic cleavage, heterolytic cleavage, and hydrogen-shift rearrangements) are introduced in a clear manner, then supported systematically with nearly 100 fragmentation schemes to facilitate interpretation of dozens of representative mass spectra of various types of compounds. This chapter also includes detailed information on EI mass spectral databases and library search programs along with descriptions of their use. The basis for chemical ionization is described in Chapter 7. Whereas positive-ion formation is emphasized, attention is also given to negative-ion formation, with careful distinction between negative-ion CI (NCI, the result of an ion/molecule reaction involving an anion) and electron capture negative ionization (ECNI), a resonant process involving capture of a thermal electron. Atmospheric pressure CI (APCI) is introduced, which serves as an important interface for LC/MS applications that are not amenable to electrospray ionization. The specialized technique of desorption CI (DCI) is also described. Descriptions of the various types of CI are supported with illustrative examples of application to environmental and biomedical problems. The operating principles of electrospray ionization (ESI) are described in Chapter 8 together with some of the mechanical aspects of the interface that make it one of the most viable for LC/MS applications. The basis for automated computation of the mass of the analyte is illustrated in an example that dissects the peaks in an ESI mass spectrum and sets up simultaneous equations based on first principles relating to the m/z value of the mass spectral peaks. Although introduced in Chapter 4, the developing technique of DESI is covered. Many current applications of the ESI technology are reviewed, which results in more than 300 references in this chapter. The operating principles of matrix-assisted laser desorption/ionization (MALDI) are described in Chapter 9, including some commentary on current theories of the mechanism of ionization. Attention is given to sample preparation, including descriptions of specialized sample probes to facilitate sample cleanup. Examples of typical MALDI spectra are described to illustrate the effect and use of delayed extraction and ion mirrors (reflectrons). The technology of atmospheric pressure MALDI (AP MALDI) is described. Many current applications of MALDI technology are reviewed, also making this chapter rich in citations (more than 500). Chapter 10 describes the basis for trade-offs in individual operation of GC and MS that are necessary for successful operation of the combined technique. Introductory protocols for proper syringe/sample handling in the mature technology of GC/MS are presented. The important technology of selected ion monitoring (SIM) is described in the context of qualitative and quantitative applications in the biomedical and environmental fields. Strategies and procedures for data processing with mass chromatograms are described in the context of suspected overlapping data obtained from samples containing chromatographically unresolved components. Some current applications of the technology are reviewed along with explanations of software used for component deconvolution through processing complex data. This chapter has nearly 200 references. xx

23 Preface Chapter 11 on LC/MS emphasizes how conventional protocols of HPLC operation must be modified to become compatible with MS operation for combined operation. Although electrospray is the dominant interface for LC/MS, the specialized ionization techniques of APCI and APPI (atmospheric pressure photoionization), which lend themselves to particular applications, are given serious consideration. Several current applications of LC/MS technology are reviewed resulting in almost 250 citations. Methodology for proteomics is emphasized in Chapter 12, which also describes some basic approaches to the characterization of carbohydrates and nucleotides. The strategy and procedure for sequencing a peptide from CAD MS/MS data are described in detail as supported by results for a simple didactic example. The concept of peptide mass mapping is described, which is the basis, sometimes in combination with data from CAD MS/MS, for automated identification of proteins by software that is often purchased as part of a data system or that is used in conjunction with notable Web sites for such purposes. Methodology for identifying/characterizing a variety of post-translational modifications to proteins, including phosphorylation and disulfide-bond formation, is described in the context of several step-by-step examples. Hundreds of current applications are reviewed, bringing the number of references in this chapter to more than 800. Because the book is designed for use as a textbook for courses on mass spectrometry, Power Point presentations, including figures from the book and animations developed by the authors, are available for downloading to site-registered instructors to support their teaching efforts. For the benefit of students, the authors will maintain a Web site (through and with the support of the publisher) that will contain exercises together with downloadable answer keys. These materials will be updated on a regular basis. xxi

24 Preface Determination is often the first chapter in the book of excellence. ~Unknown xxii

25 ACKNOWLEDGMENTS Many of the realistic examples of mass spectral data and applications of mass spectrometry derive from experiments conducted in the Watson Laboratory by some 50 Ph.D. graduate students or postdoctoral fellows in the context of biomedical research applications. Recent contributors include graduate assistants Xue Li, Jose-Luis Gallegos-Perez, Nalini Sadagopan, Naxing Xu, Yingda Xu, Wei Wu, Jianfeng Qi, David Wagner, and professorial students Heidi Bonta, Brad Sauter, and Greg Boyd. Other illustrative applications of mass spectrometry derive from the Sparkman Laboratory, with the help of Teresa Vail and Matthew Curtis, in the context of environmental chemistry and computational approaches to preparing and interrogating standard libraries of mass spectra. Thanks to Leslie Behm and Susan Kendall in the MSU Library System for assistance and counsel to JTW in dealing with the vagaries of the EndNote algorithm. The integrity of information and data interpretation contained herein has been bolstered by critiques from prominent colleagues in the field, including Professors Gavin Reid, John Allison, Jack Holland, Vernon Reinhold, Robert Brown, J.A. McCloskey, A. Daniel Jones, and Drs. Christian Rolando, J. Lemoine, Steven Pomerantz, Charles Ngowe, Chad Borges, Robin Hood, John Stults, and J. David Pinkston. A special thanks to Professor Jean-Francois Gal, who generously provided lab/office space for JTW at the University of Nice for his sabbatical leave in 2002 during the formative stages of this project. The logical and systematic approach to presenting scientific/technical information that JTW learned from his mentor, Professor Klaus Biemann at MIT, was of continuing benefit during this project, and some of the critique/suggestions by Dr. Brian Sweetman and Professor John Oates at Vanderbilt University during preparation of the first edition of the book survive in this fourth edition. Thanks also to Patrick R. Jones, ODS s colleague at the University of the Pacific, for great discussions and reflections especially on instrumentation and physical chemisty. We appreciate the counsel of Frederick E. Klink, who has been our co-instructor in short courses for the last 10 years, and who has greatly added to the portions of this book involving HPLC and LC/MS. We also appreciate the cooperation of Harold G. Walsh, Director of the ACS Short Course Program, during our tenure from 1978 to 2006 with his program, and to the thousands of students who have participated in these short courses as well as the instrument manufacturers who provided equipment and other support for our hands-on courses. Special thanks go to Stephen E. Stein at the Mass Spectrometry Data Center of the National Institute of Standards and Technology for permission to use many of the EI mass spectra, which come from the NIST05 NIST/EPA/NIH Mass Spectral Database. Unless otherwise designated, spectra were taken from the NIST Mass Spectral Database. Also, the NIST Mass Spectral Search Program proved invaluable in the preparation of many of the non-ei mass spectra contained in this book. Both authors offer a special thanks to ODS s wife, Joan A. Sparkman, who spent a great number of hours implementing the suggestions of the Wiley contract-copyeditor and making sure that there was consistency throughout the book. Because of the sometimes orthogonally opposed styles of the authors, the inputs of the copyeditor and the proofreader, and further complications with the delivery of camera-ready copy, Joan has paraphrased the title of a song, saying that the style of this book can be considered a little bit country, a little bit rock 'n roll. Thanks also goes to those two great canine mass spectrometrists, Maggie and Chili Sparkman, who endured the final edits with ODS and Joan. xxiii

26 I feel sure that there are many problems in Chemistry which could be solved with far greater ease by the application of Positive Rays to chemical analysis than by any other method. ~Joseph John Thomson xxiv

27 Chapter 1 Chapter 1 Introduction I. Introduction The Tools and Data of Mass Spectrometry 2. The Concept of Mass Spectrometry II. History...9 III. Some Important Terminology Used in Mass Spectrometry Introduction 2. Ions 3. Peaks 4. Resolution and Resolving Power IV. Applications Example 1-1: Interpretation of Fragmentation Patterns (Mass Spectra) to Distinguish Positional Isomers 2. Example 1-2: Drug Overdose: Use of GC/MS to Identify a Drug Metabolite 3. Example 1-3: Verification that the Proper Derivative of the Compound of Interest Has Been Prepared 4. Example 1-4: Use of a CI Mass Spectrum to Complement an EI Mass Spectrum 5. Example 1-5: Use of Exact Mass Measurements to Identify Analytes According to Elemental Composition 6. Example 1-6: Is This Protein Phosphorylated? If So, Where? 7. Example 1-7: Clinical Diagnostic Tests Based on Quantitation of Stable Isotopes by Mass Spectrometry in Lieu of Radioactivity V. The Need for Chromatography...42 VI. Closing Remarks...44 VII. Monographs on Mass Spectrometry Published Before Introduction to Mass Spectrometry, 4th Edition: Instrumentation, Applications, and Strategies for Data Interpretation; J.T. Watson and O.D. Sparkman, 2007, John Wiley & Sons, Ltd

28 2 Introduction Fixed gases or volatile liquids by EI, CI, or FI Solids or solutions by ESI, APCI, AP-MALDI Ion Source IONS m/z Analyzer Ions Detector Solids by MALDI or LSIMS Vacuum Computer/data system 1 Figure 1. This conceptual illustration of the mass spectrometer shows the major components of mass spectrometer, i.e., sample inlets (dependent on sample and ionization technique; ion source (origin of gas phase ions); m/z analyzer (portion of instrument responsible for separation of ions according to their individual m/z values); detector (generates the signals that are a recording of the m/z values and abundances of the ions); vacuum system (the components that remove molecules, thereby providing a collision-free path for the ions from the ion source to the detector); and the computer (coordinates the functions of the individual components and records and stores the data).

29 Introduction 3 I. Introduction Mass spectrometry is a microanalytical technique that can be used selectively to detect and determine the amount of a given analyte. Mass spectrometry is also used to determine the elemental composition and some aspects of the molecular structure of an analyte. These tasks are accomplished through the experimental measurement of the mass of gas-phase ions produced from molecules of an analyte. Unique features of mass spectrometry include its capacity for direct determination of the nominal mass (and in some cases, the molar mass) of an analyte, and to produce and detect fragments of the molecule that correspond to discrete groups of atoms of different elements that reveal structural features. Mass spectrometry has the capacity to generate more structural information per unit quantity of an analyte than can be determined by any other analytical technique. Much of mass spectrometry concerns itself with the mass of the isotopes of the elements, not the atomic mass 1 of the elements. The atomic mass of an element is the weighted average of the naturally occurring stable isotopes that comprise the element. Mass spectrometry does not directly determine mass; it determines the mass-to-charge ratio (m/z) of ions. More detailed explanations of atomic mass and mass-to-charge ratios follow in this chapter. It is a fundamental requirement of mass spectrometry that the ions be in the gas phase before they can be separated according to their individual m/z values and detected. Prior to 1970, only analytes having significant vapor pressure were amenable to mass spectrometry because gas-phase ions could only be produced from gas-phase molecules by the techniques of electron ionization (EI) or chemical ionization (CI). Nonvolatile and thermally labile molecules were not amenable to these otherwise still-valuable gas-phase ionization techniques. EI (Chapter 6) and CI (Chapter 7) continue to play very important roles in the combined techniques of gas chromatography/mass spectrometry (GC/MS, Chapter 10) and liquid chromatography/mass spectrometry (LC/MS, Chapter 11). After 1970, the capabilities of mass spectrometry were expanded by the development of desorption/ionization (D/I) techniques, the generic process of generating gas-phase ions directly from a sample in the condensed phase. The first viable and widely accepted technique 2 for D/I was fast atom bombardment (FAB), which required nanomoles of analyte to produce an interpretable mass spectrum. During the 1980s, electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI) eclipsed FAB, in part because they required only picomoles of analyte for analysis. ESI and MALDI are mainly responsible for the dominant role of mass spectrometry in the biological sciences today because they are suitable for analysis of femtomole quantities of thermally labile and nonvolatile analytes; therefore, a chapter is devoted to each of these techniques (Chapters 8 and 9). Mass spectrometry is not limited to analyses of organic molecules; it can be used for the detection of any element that can be ionized. For example, mass spectrometry can analyze silicon wafers to determine the presence of lead and iron, either of which can 1 In the United States, the term atomic weight is used for the relative mass of the elements. In the rest of the world, which is based on the metric system, the term atomic mass is used. This book uses the term atomic mass instead of the more widely accepted term in the U.S., atomic weight. 2 It should be mentioned that the techniques of 252 Cf (Ron MacFarlane) and Laser Microprobe Mass Analysis (LAMMA) (Franz Hillenkamp and Michael Karas) were less popular D/I techniques that were developed in the same temporal arena as FAB, but they were not commercially viable. More information on these two techniques can be found in Chapter 9.

30 4 Introduction cause failure of a semiconductor for microprocessors; similarly, drinking water can be analyzed for arsenic, which may have health ramifications. Mass spectrometry is extensively used in geology and material sciences. Each of these two disciplines has developed unique analytical capabilities for the mass spectrometer: isotope ratio mass spectrometry (IRMS) in geology and secondary ion mass spectrometry (SIMS) in material sciences. Both of these techniques, along with the analysis of inorganic ions, are beyond the scope of this present book, which concentrates on the mass spectrometry of organic substances. 1. The Tools and Data of Mass Spectrometry The tools of mass spectrometry are mass spectrometers, and the data are mass spectra. Figure 1-1 is a conceptual representation of a mass spectrometer. Each of the individual components of the instrument will be covered at logical stages throughout this book. Figure 1-2 depicts the three ways of displaying the data recorded by the mass spectrometer. The acquired mass spectra can be displayed in many different ways, which allow the desired information about the analyte to be easily extracted. These various techniques for data display and their utility are covered later in this chapter. 2. The Concept of Mass Spectrometry Ions are charged particles and, as such, their position in space can be manipulated with the use of electric and magnetic fields. When only individual ions are present, they can be grouped according to their unique properties (mass and the number of charges) and moved from one point to another. In order to have individual ions free from any other forms of matter, it is necessary to analyze them in a vacuum. This means that the ions must be in the gas phase. Mass spectrometry takes advantage of ions in the gas phase at low pressures to separate and detect them according to their mass-to-charge ratio (m/z) the mass of the ion on the atomic scale divided by the number of charges that the ion possesses. This definition of the term m/z is important to an understanding of mass spectrometry. It should be noted that the m/z value is a dimensionless number. The m/z term is always used as an adjective; e.g., the ions with m/z 256, or the ion has an m/z value of 256. A recording of the number of ions (abundance) of a given m/z value as a function of the m/z value is a mass spectrum. Only ions are detected in mass spectrometry. Any particles that are not ionic (molecules or radicals 3 ) are removed from the mass spectrometer by the continuous pumping that maintains the vacuum. The mass component that makes up the dimensionless m/z unit is based on an atomic scale rather than the physical scale normally considered as mass. Whereas the mass physical scale is defined as one kilogram being the mass of one liter of water at a specific temperature and pressure, the atomic mass scale is defined based on a fraction of a specific isotope of carbon; i.e., 1 mass unit on an atomic scale is equal to 1/12 the mass of the most abundant naturally occurring stable isotope of carbon, 12 C. This definition of mass, as represented by the symbol u, which is synonymous with dalton (Da), will be used throughout this book [1]. A previous standard for the atomic mass unit was established in chemistry in 1905 (based on the earlier suggestion of the Belgium chemist, Jean Servais 3 Both molecules and radicals are particles that have no charge. Molecules are characterized by an even number of electrons and radicals by an odd number of electrons.