METHODS IN ENZYMOLOGY EDITORS-IN-CHIEF John N. Abelson Melvin I. Simon DIVISION OF BIOLOGY CALIFORNIA INSTITUTE OF TECHNOLOGY PASADENA, CALIFORNIA FOUNDING EDITORS Sidney P. Colowick and Nathan O. Kaplan Preface Mass spectrometry deals with the formation, manipulation, and measurement of charged substances in order to detect and identify them. Since its previous overview was published in this series (McCloskey, 1990), the Nobel Prize in Chemistry was awarded in 2002 to John B. Fenn and Koichi Tanaka for the discovery of two new methods for producing charged biomacromolecules from liquid and solid solution. These can be thought of as ways to isolate charged molecules in the gas phase that are formed simply from ‘‘normal acid‐base protonation‐deprotonation reactions’’ from volatile liquid buffers and solid matrices. Over the past two decades these techniques, electrospray (ESI) and matrix‐assisted laser desorption (MALDI), have provided the remarkable window we needed to ‘‘see’’ into the machinery of cell biology and view its true molecular complexity for the first time. These ways of producing ions work efficiently for virtually all biomacromo- lecules, so it is left to our scientific ingenuity to design ways to manipulate these charged molecules to elicit information that reveals their molecular structural nature. Hence, several generations of ion‐optical and energy deposition strate- gies have emerged that make up the current tools of the trade—commercial mass spectrometers. It should be noted that the design and discovery of better strategies remains a vibrant, young pursuit. Finally, advanced computational capabilities have evolved to record, pro- cess, and manage mass spectral information and provide interfaces with DNA and protein sequence repositories. The tools of bioinformatics are also being adapted and refined to provide visualization into our existing knowledge of biology. But these developments represent just the beginning of positioning the kind of ingredients that will be employed to gain an understanding of human biology. This volume and its companion (Burlingame, 2005) are intended to describe the astounding strides that have brought us to our current methodological toolbox and also provide the foundation of knowledge indispensable to under- standing the current practice of mass spectrometry, as well as to appreciate the rapidly expanding and accelerating horizons in this field. Thus, this work is focused at the forefront of proteins and their complexities, including descriptions of the techniques and instrumentation being used, their sequence and structural identification based on interpretation of their tandem mass spectra, the strategies and issues in proteomics, studies of solution ix x preface structures and interactions using isotope exchange, studies of non‐covalent complexes with metal ions and ligands, and use of sub‐attomole isotopic bio‐ tracers using accelerator mass spectrometry. All of these contributions are written by authorities who have made seminal contributions to their respective topics. These foundations provide insight into the forefront of the experimental and technological platforms necessary to pursue a variety of major research themes surrounding protein biology, including proteomics, protein‐protein interac- tions, glycobiology, epi‐genetics, and systems biology. I am indebted to all of my colleagues who have participated in this work, to Candy Stoner for her assistance and talents during the preparation phase, and to Raisa Talroze for the completion of both volumes. I would like to acknowl- edge the NIH, National Center for Research Resources, for generous financial support (Grant RR 01614). A. L. BURLINGAME References Burlingame, A. L. (2005). ‘‘Mass Spectrometry: Modified Proteins and Glycoconjugates.’’ Meth. Enz. 405. McCloskey, J. A. (1990). Mass spectrometry. Meth. Enz. 193, 960. Table of Contents CONTRIBUTORS TO VOLUME 402 . . . . . . . . . . . . . . . . . vii PREFACE . . . . . . . . . . . . . . . . . . . . . . . . ix VOLUMES IN SERIES . . . . . . . . . . . . . . . . . . . . . xi Biological Mass Spectrometry 1. Mass Spectrometers for the Analysis of MICHAEL A. BALDWIN 3 Biomolecules 2. Hybrid Quadrupole/Time-of-Flight Mass WERNER ENS AND Spectrometers for Analysis of Biomolecules KENNETH G. STANDING 49 3. Tandem Time-of-Flight Mass Spectrometry MARVIN L. VESTAL AND JENNIFER M. CAMPBELL 79 4. Tandem Mass Spectrometry in Quadrupole ANNE H. PAYNE AND Ion Trap and Ion Cyclotron Resonance GARY L. GLISH 109 Mass Spectrometers 5. Collision-Induced Dissociation (CID) of J. MITCHELL WELLS AND Peptides and Proteins SCOTT A. MCLUCKEY 148 6. Peptide Sequencing by MALDI JOSEPH W. MORGAN, 193-nm Photodissociation TOF MS JUSTIN M. HETTICK, AND DAVID H. RUSSELL 186 7. Peptide Sequence Analysis KATALIN F. MEDZIHRADSZKY 209 8. Proteomics JOHN T. STULTS AND DAVID ARNOTT 245 9. Bioinformatic Methods to Exploit Mass ROBERT J. CHALKLEY, Spectrometric Data for Proteomic KIRK C. HANSEN, AND Applications MICHAEL A. BALDWIN 289 10. Protein Conformations, Interactions, and CLAUDIA S. MAIER AND H/D Exchange MAX L. DEINZER 312 11. Ligand–Metal Ion Binding to Proteins: NOELLE POTIER, Investigation by ESI Mass Spectrometry HE´ LE` NE ROGNIAUX, GUILLAUME CHEVREUX, AND ALAIN VAN DORSSELAER 361 v vi table of contents 12. Site-Specific Hydrogen Exchange of Proteins: ZHONG-PING YAO, Insights into the Structures of PAULA TITO, AND Amyloidogenic Intermediates CAROL V. ROBINSON 389 13. Quantitating Isotopic Molecular Labels with JOHN S. VOGEL AND Accelerator Mass Spectrometrty ADAM H. LOVE 402 14. Accelerator Mass Spectrometry for KAREN BROWN, Biomedical Research KAREN H. DINGLEY, AND KENNETH W. TURTELTAUB 423 AUTHOR INDEX . . . . . . . . . . . . . . . . . . . . . . 445 SUBJECT INDEX . . . . . . . . . . . . . . . . . . . . . . 471 Contributors to Volume 402 Article numbers are in parantheses following the name of Contributors. Affiliations listed are current. DAVID ARNOTT (8), Department of Protein KIRK C. HANSEN (9), School of Medicine, Chemistry, Genetech, Inc., South San University of Colorado Health Sciences Francisco, California Center, Aurora, Colorado MICHAEL A. BALDWIN (1, 9), Mass Spectro- JUSTIN M. HETTICK (6), National Institute metry Research Resource, Department for Occupational Safety and Health, of Pharmaceutical Chemistry, Univer- Health Effects Laboratory Division, sity of California, San Francisco, San Allergy and Clinical Immunology Francisco, California Research, Washington, D.C. KAREN BROWN* (14), Lawrence Liver- ADAM H. LOVE (13), Center for Accelera- more National Laboratory, Livermore, tor Mass Spectrometry, Lawrence Liver- California more National Laboratory, Livermore, California JENNIFER M. CAMPBELL (3), Applied Bio- systems, Framingham, Massachusetts CLAUDIA S. MAIER (10), Department of Chemistry, Oregon State University, ROBERT J. CHALKLEY (9), Department of Corvallis, Oregon Pharmaceutical Chemistry, University of California, San Francisco, San Fran- SCOTT A. MCLUCKEY (5), Department of cisco, California Chemistry, Purdue University, West La- fayette, Indiana GUILLAUME CHEVREUX (11), Laboratoire de Spe´ctrometrie de Masse Bio-Organi- KATALIN F. MEDZIHRADSZKY (7), De- que (LSMBO), Strasbourg, France partment of Pharmaceutical Chemistry, School of Pharmacy, University of Cali- MAX L. DEINZER (10), Department of fornia, San Francisco, San Francisco, Chemistry, Oregon State University, California; Proteomics Research Group, Corvallis, Oregon Biological Research Center, Szeged, Hungary KAREN H. DINGLEY (14), Lawrence Liver- more National Laboratory, Livermore, JOSEPH W. MORGAN (6), Department California of Chemistry, Texas A&M University, College Station, Texas WERNER ENS (2), Department of Physics and Astronomy, University of Manitoba, ANNE H. PAYNE (4), Department of Chem- Winnipeg, Manitoba, Canada istry, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina GARY L. GLISH (4), Department of Chem- istry, University of North Carolina NOELLE POTIER (11), Laboratoire de Spe´c- at Chapel Hill, Chapel Hill, North trometrie de Masse Bio-Organique, Carolina Strasbourg, France *Current address: Cancer Biomarkers and Prevention Group, The Biocentre, University of Leicester, Leicester, United Kingdom. vii viii contributors to volume 402 CAROL V. ROBINSON (12), Department of KENNETH W. TURTELTAUB (14), Biology Chemistry, University of Cambridge, and Biotechnology Research Program, Cambridge, United Kingdom Lawrence Livermore National Labora- tory, Livermore, California HE´ LE` NE ROGNIAUX (11), INRA URPVI – ALAIN VAN DORSSELAER (11), Laboratoire Plate-forme de Spectrome´trie de Masse, de Spe´ctrometrie de Masse Bio-Organi- Nantes, France que, Strasbourg, France DAVID H. RUSSELL (6), Department MARVIN L. VESTAL (3), Applied Bio- of Chemistry, Texas A&M University, systems, Framingham, Massachusetts College Station, Texas JOHN S. VOGEL (13), Center for Accelerator KENNETH G. STANDING (2), Department Mass Spectrometry, Lawrence Liver- of Physics and Astronomy, University more National Laboratory, Livermore, of Manitoba, Winnipeg, Manitoba, California Canada MITCHELL J. WELLS (5), Department JOHN T. STULTS (8), Predicant Biosciences, of Chemistry, Purdue University, West Inc., South San Francisco, California Lafayette, Indiana PAULATITO (12), Department of Chemistry, ZHONG-PING YAO (12), Department of University of Cambridge, Cambridge, Chemistry, University of Cambridge, United Kingdom Cambridge, United Kingdom [1] mass spectrometers for biomolecular analysis 3 [1] Mass Spectrometers for the Analysis of Biomolecules By MICHAEL A. BALDWIN Abstract Mass spectrometry (MS) has become a vital enabling technology in the life sciences. This chapter summarizes the fundamental aspects of MS, with reference to topics such as isotopic abundance and accurate mass and resolution. A broad and comprehensive overview of the instrumenta- tion, techniques, and methods required for the analysis of biomolecules is presented. Emphasis is placed on describing the soft ionization meth- ods and separation techniques employed in current state‐of‐the‐art mass spectrometers. As defined in a publication from the International Union of Pure and Applied Chemistry (IUPAC), MS (or mass spectroscopy) is ‘‘the study of systems by the formation of gaseous ions, with or without fragmentation, which are then characterized by their mass‐to‐charge ratios and relative abundances’’ (Todd, 1991). Since the publication of the last volume in Methods in Enzymology reviewing MS of biomolecules (McCloskey, 1990), there has been a revolution in the field. Two promising novel soft ionization methods emerging at that time were not generally available, partly because both were largely incompatible with the typical commercial sector mass spectrometers that were in widespread use. Although the particle bombardment/desorption techniques of plasma desorption MS (PDMS), fast atom bombardment (FAB), and liquid secondary ion MS (LSIMS), invented a decade earlier, had been making valuable contribu- tions to the analysis of peptides, oligosaccharides, and other polar and involatile compounds, they were largely limited to the picomole range and thus lacked the sensitivity needed to tackle the most challenging problems. During that period when analysis of intact biological molecules such as small proteins first became possible, much research was focused on attempts to ionize ever larger molecules, many of which were standards purchased from commercial suppliers. With hindsight, simply measuring the molecular weight of a large molecule is often of limited utility, whereas digesting it chemically or enzymatically to smaller moieties and measuring the masses of even a subset of these can be very informative. Today, thanks to the maturation of soft ionization methods and new developments in mass analyzers optimized for these new ionization methods, MS is METHODS IN ENZYMOLOGY, VOL. 402 0076-6879/05 $35.00 Copyright 2005, Elsevier Inc. All rights reserved. DOI: 10.1016/S0076-6879(05)02001-X 4 biological mass spectrometry [1] established as a fundamental technology in the biological sciences in rou- tine use in numerous laboratories worldwide. There is no doubt that it is contributing to the solution of very many fundamental problems in biology and medicine (Burlingame et al., 2000; Weston and Hood, 2004). The selection of an optimal mass spectrometric method to tackle a particular task is rarely a straightforward consideration. As an example, MS is the enabling technology in the field of proteomics (deHoog andMann, 2004), which involves protein identification in very complexmixtures such as cell lysates, tissues, or other biological samples, as well as the identification of interacting partners (Deshaies et al., 2002), characterization of modifica- tions, quantitation of expression levels, and studies of non‐covalent protein complexes. In most cases, this involves an initial separation step, usually by one‐dimensional (1D) or two‐dimensional (2D) gel electrophoresis, perhaps labeling with an affinity‐tagged ligand, followed by proteolysis with a site‐ specific protease such as trypsin to generate peptides, possibly multiple further separation/enrichment steps, then the mass spectrometric analysis of the peptide mixtures. Some of the questions that arise in choosing a mass spectrometer to carry out various aspects of these tasks are as follows: What level of sensitivity is required? How accurate must molecular weight measurements be, and is there a limit to the accuracy that is required or even useful? Will the determination of peptide molecular weight values be sufficient or will sequence data be necessary, and if so, what are the optimal techniques? Is it better to separate the peptide mixtures before MS analysis, for example, by high‐performance liquid chromatography (HPLC)‐MS, or can sufficient information be obtained on unseparated mixtures? If HPLC is not used, how will the selected method be affected by impurities that may be difficult to remove? What is the sample throughput, and is the technique amenable to automation? What will it cost? Although such questions help to narrow the choices, ultimately there will be a number of alternative solutions, each of which has individual strengths and weaknesses. Some Definitions and Principles In addition to the 1991 IUPAC recommendations on nomenclature and definitions (Todd, 1991), in the same year the Committee on Measure- ments and Standards of the American Society for Mass Spectrometry also