Introduction to Mass Spectrometry Instrumentation, Applications

and Strategies for Data Interpretation

Fourth Edition

John Wiley & Sons, Ltd

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

vi

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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 978-0-470-51634-8 (cloth) 1. Mass spectrometry. 2. Biomolecules--Analysis. I. Sparkman, O. David (Orrin David), 1942- II. Title. QC454.M3W38 2007 543'.65--dc22 2007024030

British Library Cataloguing in Publication Data A catalogue record for this book is available from the British Library

Typeset by the authors Printed and bound in Great Britain by Antony Rowe, Chippenham, Wiltshire

ISBN 978-0470-51634-8 (H/B)

Reprinted with corrections March 2008

vii

Contents

Preface................................................................................................... xix

Acknowledgments .............................................................................. xxiii

Chapter 1 Introduction......................................................................... 1 I. Introduction ..........................................................................................................3

1. The Tools and Data of Mass Spectrometry...............................................4 2. The Concept of Mass Spectrometry..........................................................4

II. History ...................................................................................................................9 III. Some Important Terminology Used In Mass Spectrometry...........................22

1. Introduction..............................................................................................22 2. Ions..........................................................................................................22 3. Peaks ......................................................................................................23 4. Resolution and Resolving Power.............................................................25

IV. Applications........................................................................................................28 1. Example 1-1: Interpretation of Fragmentation Patterns

(Mass Spectra) to Distinguish Positional Isomers .........................................................................28

2. Example 1-2: Drug Overdose: Use of GC/MS to Identify a Drug Metabolite ..........................................................29

3. Example 1-3: Verification that the Proper Derivative of the Compound of Interest Has Been Prepared...................32

4. Example 1-4: Use of a CI Mass Spectrum to Complement an EI Mass Spectrum.........................................................35

5. Example 1-5: Use of Exact Mass Measurements to Identify Analytes According to Elemental Composition..............37

6. Example 1-6: Is This Protein Phosphorylated? If So, Where? ...........39 7. 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 1970 ........................44

Chapter 2 The Mass Spectrometer ................................................... 53 I. Introduction ........................................................................................................55 II. Ion Guides...........................................................................................................56 III. Types of m/z Analyzers......................................................................................61

1. Time-of-Flight m/z Analyzers...................................................................62 A. Linear ................................................................................................64

1) Resolving Power of the Linear TOF Instrument .........................65 2) Time-Lag Focusing ....................................................................66 3) Beam Deflection.........................................................................67

B. Reflectron .........................................................................................69 C. Orthogonal Acceleration ...................................................................74

Contents

viii

D. Ion Detection in the TOF Analyzer....................................................75 1) Time-Slice Detection ..................................................................76 2) Time-Array Detection .................................................................77 3) TAD with Transient Recorders ...................................................79 4) TAD with an Integrating Transient Recorder ..............................79 5) Hadamard Transform TOF–MS .................................................80

2. 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.........................................100

3. The Orbitrap ..........................................................................................103 A. Historical Aspects ...........................................................................103 B. Operating Principles........................................................................103

1) Role of the C Trap in Success of the Orbitrap .........................106 2) Figures of Merit for the Orbitrap as an m/z Analyzer................107

4. Transmission Quadrupoles ...................................................................108 A. QMF Equations of Motion ...............................................................109 B. The Stability Diagram......................................................................110 C. Characteristics of Output ................................................................111 D. Spectral Skewing ............................................................................113 E. Performance Limitations .................................................................115

5. Magnetic-Sector Instruments ................................................................115 A. Single-Focusing Instruments ..........................................................116

1) Operating Principles .................................................................116 2) Magnetic Versus Scanning.......................................................117 3) Performance Limitations ..........................................................118

B. Double-Focusing Instruments.........................................................118 6. FTICR-MS .............................................................................................122

A. Hardware Configuration ..................................................................123 B. Operational Considerations ............................................................126 C. Representative Applications ...........................................................127

7. Ion Mobility Spectrometry (IMS) ............................................................128 A. Operating Principles........................................................................128 B. FAIMS.............................................................................................129 C. Applications.....................................................................................130

IV. Calibration of the m/z Scale ............................................................................131 1. Electron Ionization .................................................................................132 2. Chemical Ionization ...............................................................................133 3. Electrospray Ionization and APCI Techniques ......................................134 4. MALDI....................................................................................................135

V. Ion Detectors ....................................................................................................136 1. General Considerations.........................................................................136 2. Types of Detectors ................................................................................137

A. Faraday Cup ...................................................................................138 B. Electron Multiplier ...........................................................................139

1) Discrete-Dynode Version .........................................................139 2) Continuous-Dynode Version.....................................................140

C. Negative-Ion Detection ...................................................................142 D. Post-Acceleration Detection and Detection of High-Mass Ions ......143 E. Channel Electron Multiplier Array (CEMA)......................................144

Contents

ix

F. Electro-Optical Ion Detection ..........................................................144 G. The Daly Detector ...........................................................................145 H. Cryogenic Detectors .......................................................................146 I. Ion Detection in FTMS ....................................................................147

VI. Vacuum Systems..............................................................................................147 1. Introduction............................................................................................147 2. Definitions..............................................................................................148 3. Pressure Gauges ..................................................................................150

A. Thermal-Conductivity Gauges ........................................................150 B. Pirani Gauge...................................................................................150 C. Thermocouple Gauges ...................................................................151

4. Ionization Gauges..................................................................................151 A. Hot-Cathode Gauge........................................................................151 B. Cold-Cathode Gauge......................................................................151

5. Types of Pumps ....................................................................................152 A. Mechanical Pumps (Low Vacuum) .................................................152

1) Rotary Vane Pumps .................................................................153 2) Scroll Pumps ............................................................................154 3) Roots Pumps............................................................................155 4) Diaphram Pumps .....................................................................156

B. High Vacuum ..................................................................................156 1) Turbomolecular Pumps ............................................................156 2) Oil Diffusion Pumps..................................................................157 3) Sputter-Ion Pumps (Nonregeneratable Getter Pumps)............162

Chapter 3 Mass Spectrometry/Mass Spectrometry....................... 173 I. Introduction ......................................................................................................175

1. History and the Evolution of the Technique...........................................175 2. Concept and Definitions ........................................................................176 3. Nomenclature ........................................................................................177

II. Ion Dissociation................................................................................................179 1. Metastable Ions .....................................................................................179 2. Collisionally Activated Dissociation .......................................................180 3. Electron Capture Dissociation ...............................................................181 4. Electron-Transfer Dissociation ..............................................................181 5. Illustrative Example of Qualitative Analysis by MS/MS .........................184

III. Instrumentation for MS/MS .............................................................................184 1. Tandem-in-Space Mass Spectrometry (MS/MS)...................................186

A. Triple-Quadrupole Mass Spectrometer ..........................................187 B. Q-TOF Hybrid Mass Spectrometer .................................................188 C. TOF-TOF Mass Spectrometer........................................................190 D. BEqQ Hybrid Mass Spectrometer...................................................190 E. Double-Focusing Instrument...........................................................191

2. Tandem-in-Time Mass Spectrometry....................................................192 IV. Specialized Techniques and Applications.....................................................196

1. In-Source CAD ......................................................................................196 2. CAD in Conjunction with Soft Ionization ................................................197

A. Data-Dependent Acquisition ...........................................................199

Contents

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3. Selected Reaction Monitoring................................................................199 A. Illustrative Example Showing that SRM Has a Higher

Specificity than SIM in Spite of a Lower Signal Strength ................199 B. An Example Comparing the Specificity of SRM and SIM in the

Context of Analyzing a Biological Sample for a Drug Metabolite....201 4. Precursor-Ion Analysis ..........................................................................204 5. Neutral-Loss (Common Neutral-Loss) Analysis ....................................205 6. Ion/Molecule Reactions .........................................................................206 7. Hybrid Instrumentation for MS/MS and Ion Mobility

Spectrometry (IMS) ...............................................................................206 V. Analyte Identification from MS/MS Data ........................................................208

1. Introduction............................................................................................208 2. Identifying an Unknown Using a Product-Ion Mass Spectrum ..............209 3. Similarities between EI and Product-Ion Mass Spectra ........................214 4. Another Way of Using Substructure Identification.................................215 5. Searching of Product-Ion Spectra against Standardized Databases ....218

VI. Concluding Remarks about MS/MS................................................................220

Chapter 4 Inlet Systems .................................................................. 229 I. Introduction ......................................................................................................231 II. Batch Inlets .......................................................................................................232

1. Heated Reservoir Inlet...........................................................................232 2. Direct Inlet Probe (DIP) .........................................................................233

A. The Chromatoprobe........................................................................235 3. Direct Exposure Probe (Desorption Chemical Ionization, DCI).............236 4. Pyrolysis ................................................................................................238

III. Continuous Inlets .............................................................................................239 1. Membrane Introduction MS (MIMS) ......................................................239 2. Supercritical Fluid Chromatography (SFC) ...........................................240 3. Electrophoretic Inlet...............................................................................242

IV. Ionization Inlet Systems ..................................................................................244 1. Direct Analysis in Real Time (DART) ....................................................245 2. Desorption Electrospray Ionization (DESI) ............................................247 3. Desorption Atmospheric Pressure Chemical Ionization (DAPCI)..........249

V. Speciality Interfaces.........................................................................................249 1. Selected Ion Flow Tube Mass Spectrometry (SIFTMS)........................249 2. Fast Atom Bombardment (FAB) and Liquid Secondary Ion Mass

Spectrometry (LSIMS)...........................................................................250 3. Chemical Reaction Interface Mass Spectrometry (CRIMS) ..................252 4. Inductively Coupled Plasma Mass Spectrometry (ICPMS) ...................253

A. Hardware Configuration ..................................................................254 B. Operational Considerations ............................................................254 C. Electrothermal Vaporization............................................................255 D. Laser Ablation .................................................................................255 E. Speciation .......................................................................................256 F. Summary.........................................................................................256

VI. Final Statement.................................................................................................257

Contents

xi

Chapter 5 Strategies for Data Interpretation (Other than Fragmentation) ........................................... 267

I. Introduction ......................................................................................................269 II. Some Important Definitions ............................................................................271 III. Possible Information That Can Be Obtained from the Mass Spectrum......271 IV. Elemental Composition of an Ion and the Ratios of Its Isotope Peaks ......273

1. Definition of Terms Related to the Matter of Mass Spectrometry .........273 2. Nitrogen Rule ........................................................................................275 3. Elemental Composition of an Ion Based on the Ratio of

Isotope Peak Intensities ........................................................................276 A. Isotope Peak Patterns Used to Determine the Elemental

Composition of Ions ........................................................................276 B. Isotope Peak Patterns for Ions Containing Various

Combinations of Br/Cl.....................................................................279 C. Constraint on the Number of Atoms Allowed for a

Given Element ................................................................................281 D. Relationship of the Charge State of an Ion and the

Spacing of the Corresponding Isotope Peaks ................................281 1) Ions of High Mass-to-Charge Ratio..........................................282

E. Steps to Assigning an Elemental Composition Based on Isotope Peak Intensities .................................................283

F. Validating the Putative Elemental Composition of an Ion ...............284 G. An Illustrative Example of the Use of Isotope Peak Ratios to

Determine an Elemental Composition ............................................285 H. Potential Problems Arising from Adjacent Peaks ...........................291

4. Elemental Composition as a Function of an Accurate Determination of the m/z Value of a Mass Spectral Peak.....................293 A. Appearance of Mass Spectra of High-m/z Value Ions ....................295

5. Using EI Data to Identify Unknowns Detected During Analysis by LC/MS ................................................................................297

6. Does the Result Make Sense?..............................................................299 V. Identifying the Mass of an Analyte .................................................................302

1. Recognition of the Peak Representing the Molecular Ion in EI .............304 A. Reasonable Losses from the Molecular Ion in EI ...........................305

2. Recognition of the Protonated Molecule (MH ) in Soft Ionization..........305 A. Probable Adducts Observed in the Mass Spectrum

Produced by Soft Ionization ............................................................306 3. Recognition of the Deprotonated Molecule ([M H] ) Peak

in Soft Ionization ....................................................................................306 VI. Recognition of Spurious Peaks in the Mass Spectrum................................307

1. Noise Spikes .........................................................................................307 2. Peaks Corresponding to Contaminants in GC/MS and LC/MS.............307

A. The Phthalate Ion Peak ..................................................................307 B. GC Column Bleed...........................................................................308 C. Cluster Ions.....................................................................................308

VII. Obtaining Structural Information from the Mass Spectrum ........................308

Contents

xii

Chapter 6 Electron Ionization ......................................................... 315 I. Introduction ......................................................................................................317 II. Ionization Process............................................................................................317 III. Strategy for Data Interpretation ......................................................................321

1. Assumptions..........................................................................................321 2. The Ionization Process..........................................................................321

IV. Types of Fragmentation Pathways.................................................................328 1. Sigma-Bond Cleavage ..........................................................................330 2. Homolytic or Radical-Site-Driven Cleavage ..........................................333 3. Heterolytic or Charge-Site-Driven Cleavage .........................................335 4. Rearrangements....................................................................................337

A. Hydrogen-Shift Rearrangements ....................................................338 B. Hydride-Shift Rearrangements .......................................................342

V. Representative Fragmentations (Spectra) of Classes of Compounds...........344 1. Hydrocarbons ........................................................................................345

A. Saturated Hydrocarbons .................................................................345 1) Straight-Chain Hydrocarbons ...................................................345 2) Branched Hydrocarbons...........................................................348 3) Cyclic Hydrocarbons.................................................................351

B. Unsaturated ....................................................................................353 C. Aromatic..........................................................................................355

2. Alkyl Halides ..........................................................................................363 3. Oxygen-Containing Compounds ...........................................................368

A. Aliphatic Alcohols............................................................................368 B. Aliphatic Ethers ...............................................................................372 C. Aromatic Alcohols ...........................................................................376 D. Cyclic Ethers ...................................................................................381 E. Ketones and Aldehydes ..................................................................381 F. Aliphatic Acids and Esters ..............................................................392 G. Aromatic Acids and Esters..............................................................402

4. Nitrogen-Containing Compounds ..........................................................405 A. Aliphatic Amines .............................................................................405 B. Aromatic Compounds Containing Atoms of Nitrogen .....................414 C. Heterocyclic Nitrogen-Containing Compounds...............................419 D. Nitro Compounds............................................................................419 E. Concluding Remarks on the Mass Spectra of

Nitrogen-Containing Compounds ...................................................420 5. Multiple Heteroatoms or Heteroatoms and a Double Bond...................421 6. Trimethylsilyl Derivative.........................................................................422 7. Determining the Location of Double Bonds...........................................429

VI. Library Searches and EI Mass Spectral Databases ......................................433 1. Databases .............................................................................................433 2. Library Search Programs ......................................................................435 3. What To Do When the Spectrum of the Unknown is Not in the

Database(s)...........................................................................................439 4. Searching Multiple Databases...............................................................440 5. Database Size and Quality ....................................................................440 6. Concluding Remarks on the NIST Mass Spectral Search Program .....441

VII. Summary of Interpretation of EI Mass Spectra .............................................442

Contents

xiii

Chapter 7 Chemical Ionization........................................................ 449 I. Introduction ......................................................................................................451 II. Description of the Chemical Ionization Source.............................................454 III. Production of Reagent Ions from Various Reagent Gases..........................455 IV. Positive-Ion Formation Under CI ....................................................................457

1. Fundamentals........................................................................................457 2. Practical Consideration of Proton Affinity in CI......................................460 3. Selective Ionization................................................................................461 4. Fragmentation .......................................................................................461

V. Negative-Ion Formation under CI ...................................................................464 1. True Negative Chemical Ionization .......................................................464 2. Resonant Electron Capture Negative Ionization....................................465

VI. Data Interpretation and Systematic Studies of CI.........................................469 VII. Ionization by Charge Exchange......................................................................470

1. Mechanism of Ionization........................................................................470 2. Fragmentation and Appearance of Mass Spectra.................................471

VIII. Atmospheric Pressure Chemical Ionization ..................................................471 IX. Desorption Chemical Ionization .....................................................................472 X. General Applications........................................................................................474 XI. Concluding Remarks .......................................................................................477

Chapter 8 Electrospray Ionization .................................................. 485 I. Introduction.......................................................................................................... 487 II. Operating Principles ........................................................................................... 487 III. Appearance of ESI Mass Spectra and Data Interpretation............................. 490 IV. ESI with an m/z Analyzer of High Resolving Power........................................ 493 V. Conventional ESI Source Interface ................................................................... 494 VI. Nanoelectrospray and Microelectrospray Ionization...................................... 494 VII. Desorption Electrospray Ionization (DESI) ..................................................496 VIII. Effect of Composition and Flow Rate of an Analyte Solution ....................... 499 IX. Special Applications ........................................................................................... 500

1. Direct Analysis of Ions in Solution by ESI..............................................500 2. Cold-Spray Ionization ............................................................................501 3. Negative-Ion Detection..........................................................................501 4. Secondary Electrospray Ionization (SESI) ............................................502 5. Kinetic Measurements of Chemical Reactions......................................502 6. ESI Generation of Ions for Ancillary Experiments .................................502

X. General Applications of ESI ............................................................................... 503

Chapter 9 MALDI .............................................................................. 519 I. Historical Perspective and Introduction............................................................. 521 II. Operating Principles ............................................................................................. 521

1. The Matrix .................................................................................................. 521 2. The Laser, m/z Analyzer, and Representative Mass Spectra .................. 525 3. The Ionization Process .............................................................................. 529 4. High-Pressure (HP) MALDI and Atmospheric Pressure

(AP) MALDI................................................................................................ 533

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xiv

III. Sample Handling....................................................................................................535 1. Sample Preparation of the Conventional Plate..........................................535 2. The Problem of Analyte Solubility ..............................................................537 3. The Problem of Sample Purity...................................................................537 4. On-Probe Sample Purification and/or Modification ...............................538

A. SAMs and Polymer-Modified Surfaces ...............................................538 B. Affinity Surfaces...................................................................................540

5. Direct Analysis from Gels...........................................................................541 6. Hydrogen/Deuterium Exchange.................................................................542

IV. Special Instrumental Techniques ........................................................................542 1. Post-Source Decay (PSD) .........................................................................542 2. Ion Excitation ..............................................................................................544 3. Delayed Extraction (DE).............................................................................545 4. Desorption Ionization On Silicon (DIOS)....................................................546 5. Tissue Profiling or Imaging.........................................................................547

V. Representative Applications ................................................................................549 1. Proteins and Peptides ................................................................................549 2. Microbes .....................................................................................................549 3. Biomarkers .................................................................................................550 4. Synthetic Polymers.....................................................................................550 5. Small Molecules .........................................................................................551 6. Quantitation ................................................................................................552 7. Combined with Liquid Chromatography.....................................................553

Chapter 10 Gas Chromatography/Mass Spectrometry................... 571 I. Introduction ......................................................................................................573 II. Introduction to GC............................................................................................575

1. Basic Types of Injectors ........................................................................582 2. Injection Considerations and Syringe Handling.....................................583 3. Syringeless Modes of Sample Injection for Fast GC.............................585

III. Sample Handling ..............................................................................................585 1. Proper Sample Container......................................................................585 2. Analyte Isolation and Purification ..........................................................587 3. Derivative Formation .............................................................................587

A. Silyl Derivatives...............................................................................588 B. Esters of Carboxylic Acids ..............................................................588 C. Oxime Derivatives...........................................................................589 D. Acyl Derivatives...............................................................................590 E. Derivatives for Characterizing Double Bonds .................................590

IV. Instrument Requirements for GC/MS .............................................................590 1. Operating Pressures .............................................................................590 2. Typical Parameters for a Conventional GC-MS Interface .....................593 3. Supersonic Molecular Beam Interface for GC/MS ................................594 4. Open-Split Interface ..............................................................................596 5. Molecular Separators ............................................................................597

A. Jet-Orifice Separator.......................................................................597 B. Membrane Separator......................................................................598

6. Inertness of Materials in the Interface ...................................................599

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xv

V. Operational Considerations ............................................................................601 1. Spectral Skewing...................................................................................601 2. Background/Bleed .................................................................................602 3. The Need for Rapid Acquisition of Mass Spectra .................................604

A. Performance Trade-Offs of Conventional Instruments for GC/MS...................................................................605

B. Time-Array Detection......................................................................605 4. Selected Ion Monitoring (SIM)...............................................................606

A. Definition and Nomenclature ..........................................................606 B. Development of the Technique.......................................................607 C. Qualitative Example of SIM ............................................................607 D. Quantitative Example of SIM ..........................................................609 E. Mechanics of Ion Monitoring...........................................................613

1) Adjustment of the Mass Scale..................................................613 2) Mass Range .............................................................................613 3) Magnetic Mass Spectrometer ..................................................613 4) Transmission Quadrupole Mass Spectrometer........................614 5) Number of Ion Currents (Masses)............................................614

F. Programmable SIM.........................................................................614 G. SIM at High Resolving Power .........................................................615

VI. Sources of Error ...............................................................................................616 1. Errors Relating to Equipment or Procedure ..........................................616 2. Errors Relating to Contamination ..........................................................617 3. Sources of Interference.........................................................................618 4. Dealing with Background in a Mass Spectrum......................................618

A. AMDIS (Automated Mass spectral Deconvolution and Identification System)...............................................................622

B. Other Software Techniques ............................................................629 VII. Representative Applications of GC/MS..........................................................631 VIII. Special Techniques..........................................................................................631

1. Purge and Trap .....................................................................................631 2. Thermal Desorption...............................................................................632

Chapter 11 Liquid Chromatography/Mass Spectrometry ............... 639 I. Introduction .....................................................................................................641 II. Historical Milestones in the Development of the Interface ..........................642

1. Introduction............................................................................................642 2. The Direct Inlet ......................................................................................642 3. The Moving-Belt Interface .....................................................................644 4. The Thermospray Interface...................................................................644 5. Continuous-Flow FAB ...........................................................................646

III. Currently Viable Versions of the Interface.....................................................647 1. Atmospheric Pressure Ionization...........................................................647

A. Electrospray Ionization Interface.....................................................647 1) Optimization for Analyses by HPLC .........................................648 2) Capillary Electrophoresis Interface...........................................650

B. APCI Interface ................................................................................650

Contents

xvi

C. APPI Interface.................................................................................654 1) Operating Principles of APPI....................................................654 2) Operating Mechanics for APPI .................................................656 3) Signal Suppression ..................................................................657 4) Applications of APPI.................................................................658

2. Particle Beam Interface.........................................................................659 3. Electron Ionization and LC/MS..............................................................661

IV. Special Operation of LC under MS Conditions .............................................661 1. Effects of Mobile-Phase Composition ...................................................661

A. Signal Suppression .........................................................................662 B. Use of Internal Standards in the Face of

Signal Suppression .........................................................................663 C. Adjusting the Chromatography in the Face of Signal

Suppression during LC/MS.............................................................663 D. Ion Pairing and Signal Suppression................................................663 E. Influence of the Type and the Nature of LC Buffer .........................665 F. Influence of Solvent Composition on the ESI Signal.......................665 G. Adduct Formation ...........................................................................667 H. Spectral Interference ......................................................................668 I. System Compromise ......................................................................668

2. Differences in Method Development for ESI vs APCI ...........................672 V. Applications ......................................................................................................674

1. Attention to High Throughput ................................................................676

Chapter 12 Analysis of Proteins and Other Biopolymers .............. 689 I. Introduction ......................................................................................................691 II. Proteins .............................................................................................................691

1. Sequencing............................................................................................693 A. Nomenclature and Fragmentation in Sequencing of Peptides .......693

1) Nomenclature ...........................................................................693 2) Fragmentation ..........................................................................695

B. Strategy for Deducing Amino Acid Sequence via CAD of Peptides ......................................................................................701 1) An Illustrative Example.............................................................702 2) Possible Pitfalls in Interpretation ..............................................705 3) Search for Confirming Ions ......................................................706 4) Ladder Sequencing ..................................................................707

2. Mass Mapping .......................................................................................709 A. Peptide Mass Fingerprinting ...........................................................709 B. De novo Sequencing.......................................................................710 C. Sequence Tagging..........................................................................710 D. Sequest...........................................................................................710 E. Evaluation of Hits in Automated Searches .....................................711 F. Data-Dependent Analysis by Mass Spectrometry ..........................712

3. Post-Translational Modifications ...........................................................712 A. Recognition of Sites of Protein Phosphorylation.............................714

1) An Illustrative Example.............................................................715 2) Selective Capture and Detection of Phosphopeptides .............718 3) Chemical Modification of Phosphorylation Sites ......................719

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xvii

B. Recognition of Sites of Sulfation.....................................................722 C. Recognition of Sites of Glycosylation..............................................723 D. Acetylation of Lysine .......................................................................724 E. Cysteine Status in Proteins.............................................................726

1) Are There Any Disulfide Bonds? ..............................................726 2) Which Cysteines Are Free? .....................................................727 3) What Is the Linkage of Cysteines in the

Disulfide Bonds? ......................................................................727 (A) Conventional Proteolytic Mass Mapping

of Disulfides ......................................................................... 727 (B) Cyanylation-Based Mass Mapping of Disulfides................. 730

F. Recognition of Ubiquinated Proteins...............................................734 G. Other Types of Modifications ..........................................................735

4. Quantitation in Proteomics ....................................................................735 A. ICATs..............................................................................................735

1) Operating Principles .................................................................735 2) Illustrative Example of the ICAT Approach ..............................737 3) Analogous to ICAT Methodologies...........................................740

B. Alternative Stable Isotope-Based Methodologies ...........................740 C. Related Methodologies ...................................................................743

5. “Top-Down” Strategies of Analysis........................................................744 A. Instrumentation and Fragmentation Requirements ........................744 B. Electron Capture Dissociation (ECD) .............................................749 C. Electron-Transfer Dissociation (ETD).............................................752 D. Applications.....................................................................................753

6. Noncovalent Interactions.......................................................................753 7. Folding and Unfolding ...........................................................................756 8. Applications ...........................................................................................759

III. Oligonucleotides ..............................................................................................760 1. Analytical Considerations ......................................................................760 2. Sequencing ...........................................................................................761

A. Nomenclature .................................................................................761 B. Algorithm for Data Interpretation.....................................................763

3. Applications ...........................................................................................764 IV. Carbohydrates ..................................................................................................765

1. Analytical Considerations ......................................................................765 2. Nomenclature ........................................................................................765 3. Diagnostic Fragmentation .....................................................................766 4. Applications ...........................................................................................769

Subject Index........................................................................................ 803

Contents

xviii

xix

PREFACE

This edition of Introduction to Mass Spectrometry is far more than a revision of the third edition, which appeared in 1997. 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 2000. 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.

Preface

xx

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.

Preface

xxi

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.

Preface

xxii

“Determination is often the first chapter in the book of excellence.”

~Unknown

xxiii

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.

xxiv

“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

Chapter 1

Chapter 1 Introduction

I. Introduction ..........................................................................................................3 1. The Tools and Data of Mass Spectrometry 2. The Concept of Mass Spectrometry

II. History ...................................................................................................................9III. Some Important Terminology Used in Mass Spectrometry...........................22

1. Introduction 2. Ions 3. Peaks 4. Resolution and Resolving Power

IV. Applications........................................................................................................28 1. 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 1970 ........................44

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

2 Introduction

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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 mass1 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 ratiosfollow 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 ch rom at og r ap h y/m as s spec t rom et r y (G C/ M S, C h ap te r 1 0 ) a n d l i qu i d 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 technique2 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

1In 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.

2It should be mentioned that the techniques of 252Cf (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.

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 radicals3) 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, 12C. 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

3Both 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.