Anal. Chem. 1996, 68, 599R-651R
Mass Spectrometry A. L. Burlingame*
Department of Pharmaceutical Chemistry, The Mass Spectrometry Facility and the Liver Center, University of California, San Francisco, California 94143-0446 Robert K. Boyd
Institute for Marine Biosciences, National Research Council, Halifax, Nova Scotia, Canada B3H 3Z1 Simon J. Gaskell
Department of Chemistry, University of Manchester Institute of Science & Technology, Manchester, U.K. Review Contents Overview Scope Innovative Techniques and Instrumentation Membrane Inlet Mass Spectrometry Advances in Matrix-Assisted Laser Desorption/ Ionization Time-of-Flight Mass Spectrometry Other Developments in Time-of-Flight Mass Spectrometry Tandem Magnetic Sector/Time-of-Flight Analyzers Protein Characterization Via Database Searching Analyses of Combinatorial Chemistry Products Affinity Techniques Combined with Mass Spectrometry Electrospray Ionization at Low Flow Rates Fundamentals Ion Dissociation Dynamics Gas-Phase Ion Chromatography Gaseous Ion Thermochemistry Collisional Activation and Collision-Induced Dissociation Structures of Neutral Fragments Structures of Unusual Small Ions and Neutrals Electrochemical Aspects of Electrospray Ionization Protein Molecular Weight Determination Peptides and Proteins Synthetic Peptides Toxins, Neuropeptides, and Antigens Protein Mass Mapping Covalently Modified Protein N- and C-Termini Thiols and Disulfide Bonds Gels, Mass Mapping, Sequencing, and Database Interrogation Phosphorylation Glycosylation Lipidation Covalent Modifications Metal Ion Binding Interactions: Protein and DNA Oligonucleotides and Nucleic Acids Environmental Research and Toxicology Pollutants in the Environment Direct Detection of Genotoxicity Literature Cited
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OVERVIEW For almost a century mass spectrometry has provided key insights for an unusually broad and technically diverse suite of S0003-2700(96)00021-2 CCC: $25.00
© 1996 American Chemical Society
disciplines ranging from geochronology and space research to studies of the physical and chemical properties of materials, clusters, atoms, and particles. However, the involvement of mass spectrometry in studies of biomacromolecules stems in large part from the discovery, in 1980, that polar, labile substances could be ionized by sputtering from viscous liquid surfaces using energetic atom and ion beams. The importance of this new methodology became apparent from the variety of structural problems in protein and carbohydrate biochemistry that were tackled successfully, including de novo peptide sequence determination using tandem instrumentation. These successes led to the creation of a new disciplines biological mass spectrometryswhich addresses the challenging unsolved structural issues surrounding biopolymers of fundamental importance to the biomedical sciences. Over the last five years this expansion has accelerated dramatically, due to the discovery of further tools able to ionize larger bio-oligomers at significantly higher ionization efficiencies that also permit the detection and measurement of very large biopolymeric substances intact. These tools are electrospray (ES) (A1) and matrix-assisted laser desorption (MALD) (A2) ionization. Together with a variety of ion optical systems, they have become among the most powerful methods yet available for the macromolecular characterization of living systems (A3) and, as such, are destined to play a central role in deciphering the nature and function of the machinery of cells as we enter the post-genome era. This will lead to a detailed understanding of cell homeostasis and its modulation by extracellular communication or insult, as well as a knowledge of how protein expression and function is mediated by gene expression. Using these methods a wide range of achievements in protein research and glycobiology have been attained in a short timesin fields where earlier methods were either labor intensive or simply inadequate to fulfill needs. The unusual power of mass spectrometric techniques has been demonstrated repeatedly by the impressive results forthcoming on problems ranging from the measurement of the molecular weights and purity of large polar biopolymeric substances to delineation of the detailed sequence and structure of components of the complex mixtures obtained from their selective enzymic or chemical degradation. In addition, in the electrospray phenomenon the natural coupling of liquid chromatography and mass spectrometry was assured. As though not enough, even questions of protein folding and study of noncovalent interactions are now being tackled. Analytical Chemistry, Vol. 68, No. 12, June 15, 1996 599R
What are the general features and relative merits of these instrumental methods? What are the particularily significant developments? What does the future hold? Where are the bottlenecks? The hallmark of ionization by electrospray is formation of intact, low internal energy, even-electron ions by polyprotonation or deprotonation of neutral molecules bearing basic and/or acidic functional groups from a fine spray of their aqueous solutions (A4). Measurement of the mass values of these multiply charged species reveals the inherent molecular weights of substances as precisely as desired. However, deposition of vibronic energy is required in order to probe the structural nature of these “cold” molecular ions, that is, deposition of amounts sufficient to induce unimolecular dissociation processes (A5), thus generating a fragmentation pattern or mass spectrum. This may be carried out by a variety of instrumental techniques generically referred to as tandem mass spectrometry. In contrast, MALDI is a much more empirical technique involving laser-induced ablation of a sample as its protonated or deprotonated form from a co-crystalline solid with the matrix chosen (A6). The intent is to deposit the laser energy only into the matrix, forming a desorption/ionization plume. Thus, analyte ion formation and internal energy content varies considerably depending on the nature of the sample, the matrix chromophore and (photo)chemistry, the laser frequency and fluence, and gross variations in sample ion yield from good or less good spots on the surface holding the prepared sample (discussed in detail in other sections). Developments based on these ionization methods have been rapid and include extensive investigation of suitable methods for effective microsample handling as well as design and fabrication of new instrumentation to exploit the nature of the ionization methods and the biomacromoleules to be studied. Even the initial adaptation of these ionization techniques to existing commercial instruments over the last few years has been revolutionary in spurring research in protein biochemistry, glycobiology, and biotechnology, and most recently in opening DNA sequence analysis by detection and ordering of DNA fragments generated by Sanger dideoxy cycle sequencing (A7). But now the trend is on to more advanced instrumentation, whether the focus be on optimizing performance while lowering cost or exploring the ultimate limits. Some reports are particularily exciting, such as development of a stand alone electrospray orthogonal acceleration time-of-flight (TOF) instrument (A8), enhanced resolution, and mass measurement accuracy provided by delayed extraction MALDI-TOF (A7, A9, A10), development of the ion trap (A11, A12), and a variety of FTMS systems (A13, A14). Nevertheless, even exisiting methods and instrumentation already constitute indispensible, cost-effective tools for the rapid identification and structural characterization of biomacromolecules. The adaptation of nanoliter-per-minute flow rates to existing electrospray instruments holds considerable promise for optimization of spectral quality with concomitant minimization of sample consumption (A15, A16). Over the next few years these technologies are destined to become as commonplace in the conduct of biomedical research as the HPLC did in the early 1980s. At this juncture no one can question the pervasive scientific importance that the analytical power and versatility these methods have achieved with dazzling sensitivity and precision. On-going vigorous efforts to improve techniques to effect low-level sample 600R
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handling are now focused on achieving reliable performance in the femtomole (C80, F18, F162) to attomole ranges (A17). Development of tandem methods that will facilitate sequence determination and structual work at these levels is going on in parallel. The main unsolved problem continues to be the lack of truly effective techniques for internal energy depositionsa situation that thwarts the exploitation of tandem mass spectrometry in terms of generating complete, high-quality product ion mass spectra at ultimate sensitivities. However, despite these striking, revolutionary gains, all is not well. Formidable barriers remain to widespread use, due to a broad lack of knowledge and experience with the newest techniques and their effective uses in solving “real” biological problems, exacerbated by the lack of availability of the latest instruments in the biomedical research community. On the one hand there are too few universities that offer research opportunities and training in biological and clinical mass spectrometry, while on the other hand Congress has failed to provide adequate funding for suitable instrumentation in universities to speed biomedical research. The exception lies in the biotechnology and biopharmaceutical industry driven by potential delays in obtaining new drug approvals, when tightening regulatory requirements may not be met readily without the involvement of the best mass spectrometric methodologies. While many of the tasks in biopolymer characterization have become routine at a certain level of sensitivity (the low-picomole level) and even seem almost trivial at this stage, there are many biological problems that still present major challenges. For example, while instrumental sensitivities are certainly impressive, they are difficult to realize in many cases when working with unfamiliar biological matrices and pervasive chemical noise (A18). Very recently (A19), Zenobi et al. have investigated the MALDI matrix suppression effect in which small to moderate analyte ions (1000-20 000 Da) can fully suppress chemical noise derived from the matrix over a range of matrix to analyte mixing ratios. Interestingly, full matrix suppression can be achieved regardless of the analyte ion form (e.g., protonated or alkali adduct) and applies to all matrix species, including radical cations and proton or alkali adducts. This phenomenon is clearly of immediate practical importance but also has implications for mechanisms involved in MALDI. Indeed, Zenobi et al. (A19) proposed a model for prompt primary (not secondary gas-phase) ionization in which two excited matrix molecules are required for generation of free gaseous ions from the analyte. The model can account for the known features of the matrix suppression effect, was tested (A19) via time-delayed two-pulse MALDI experiments, and can predict the range of matrix to analyte mixing ratios for which matrix suppression is effective. It may be useful to highlight some progress from this period that indicates the current level of achievements and provides a glimpse of the future. The challenges inherent in the elucidation of protein function and macromolecular physiology in specific cell types and the availability of entire genome sequences as a giant computer look-up table is destined to involve development of separation technologies and rapid mass spectrometric-based characterization on a scale yet to be faced squarely. However the needs are clear (F148) and the opportunities abundant for both ionization techniques to play significant roles (A19, F135). There is obvious need of high-quality tandem techniques (F135) and effective database interrogation. Through use of powerful
computer algorithms, interpretation of tandem mass spectra are becoming virtually transparant for the biological scientist (A21A23). Improved strategies for studies of posttranslational and xenobiotic modifications including acylation, phosphorylation, sulfation, and glycosylation have been developed, particularly using selected ion monitoring during HPLC/ESMS analyses (A24, A25). In addition, electrospray mass spectrometry has been of particular importance in studies of heterobiopolymeric protein modifications, such as glycophosphatidylinositol membrane anchors (A26). Mass spectrometry has also been of considerable benefit together with NMR in the detailed characterization of Mycobacterial lipoarabinomannan LAM (A27), and glycolipid antigens (A28). Most recently, provocative evidence has been presented indicating that even “living” viral particles may be separated by electrospray ionization (A29). Finally, one might well ponder whether having in hand now these powerful new tools will eventually dispel the “chemical monotony of proteins and nucleic acids...” by revealing the details among their chemical forms and complexities that modulate their functions and determine their “... biological importance” (A30). SCOPE Once again the creativity and productivity of our colleagues has forced us to make some hard choices about what to omit from this review. This ever-increasing activity in mass spectrometry is exemplified by the growing literature and by the continuing growth in the numbers of presentations at the annual conferences of the American Society for Mass Spectrometry, for example. As always, we apologize in advance to those of our colleagues whose work has not received full attention in what follows. In 1996 it has become a truism (but therefore true!) to say that biological and medical applications currently represent the majority of novel developments in the discipline, and the present review again reflects this fact. At one time environmental analysis using GC/MS techniques formed one of the major areas of application of mass spectrometry, and indeed there is still a major ongoing activity in this area which has not been included in these biennial reviews for some time. The present section on Environmental Research and Toxicology contains two main thrusts, one concerned with problems of environmental science that have been successfully addressed using mass spectrometry, and the other involving more biological questions concerning mechanisms whereby harmful pollutants affect living organisms, especially via DNA modifications. Another traditional subdiscipline of mass spectrometry, which has not been covered as such in its own section for some time, is Fundamentals. After deciding that such a section should be included, we were faced with the problem of how to define it and what to include. The latter question was particularly difficult in the case of the Fundamentals section, which could easily have been 3-4 times longer than the final version, and the choice of topics included here is clearly arbitrary. This is also true of the traditional section on Innovative Techniques and Instrumentation, though in this case the coverage has been reasonably complete when averaged over the last few years, and some planned topics had to be dropped for reasons of space and deadlines. The Scope section of the 1994 edition of this review contained a polemic on the practicalities of “accurate” mass measurement and the information that can be derived from such data. This
theme is continued here with a brief overview of advances in uniquely accurate and precise measurements of atomic and molecular masses, usually undertaken by physicists in order to investigate nuclear structure, but with profound implications for some of the fundamental constants of Nature within the science of metrology. Most of the literature on this subject does not appear in journals regularly read by most mass spectrometrists. The following brief outline is intended to place the more routine “accurate mass measurements” in a broader perspective. An important point to realize from the outset is that the accuracy and precision of mass measurements in specialized ion traps is now approaching the point where chemical bond strengths, vaporization energies, etc., are significant parameters via Einstein’s famous relationship between mass and energy. That is to say, the prospect of “weighing” chemical bond strengths is now in sight (though not yet realized). As will be explained below, even now the new advances in mass measurement techniques for isolated ions are having profound implications for the determination of the Avogadro constant and definition of the mole and may even lead to an atomic standard of mass to replace the artifactual standard kilogram. The choice of mass standard for atomic and molecular masses is intimately bound up with the definition of the mole, the SI unit for amount of substance: The mole is the amount of substance of a system which contains as many elementary entities as there are atoms in 0.012 kg of carbon12. In the definition of the mole, it is understood that unbound atoms of carbon-12, at rest and in their ground state, are referred to. This wording is taken from the most recent edition of Le Syste` me Internationale d’Unite´ s (B1), published by the Bureau Internationale des Poids et Mesures (BIPM) as the internationally accredited body for metrological definitions. The number of atoms in 0.012 kg of 12C is of course the Avogadro constant (units of mol-1). The officially sanctioned symbol u for the atomic mass standard (1/12 of the mass of a single atom of carbon-12 in the gas phase (i.e., unbound), at rest and in its ground state), is not often used by chemists and biochemists, who seem to prefer to name this same unit the dalton (Da). The significance of the qualifications in the second sentence of the above definition lies in the fact that 1 nu ) 10-9 u ) 0.93 eV. Thus, if the physical state of the carbon-12 is not specified, an ambiguity arises since the heat of atomization of graphite (the thermodynamic standard state for carbon) is 7.4245 eV/atom, equivalent to 6.9 nu. As a result, 0.12 kg of 12C graphite contains about 4 × 1014 fewer atoms than the same mass of gaseous carbon atoms. Similar remarks apply to electronic excitation energy and, more importantly for mass spectrometry, to ionization energy. Thus the 12C+ ion has lost the rest mass of the electron compared with its neutral counterpart, but is also more massive by the ground-state ionization energy of the carbon atom (11.256 eV ) 10.5 nu). A discrepancy of 10 nu in the mass of a 12C atom corresponds to an uncertainty of about 1 in 109 [referred to as a part per billion (ppb) in North America but not in the U.K., where a billion is 1012]. Such uncertainties are far smaller than those achievable in routine practice by chemical mass spectrometrists. However, as pointed out by Dougherty et al. (B2), mass measurement precision of about 1 in 107 is currently achievable using FT-ICR mass spectrometers, and development of high-field instruments has the potential to push the mass measurement uncertainties down to the 1 in 109 level for chemically interesting ions. If this Analytical Chemistry, Vol. 68, No. 12, June 15, 1996
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could be achieved on a more or less routine basis, the implications for mass spectrometric determinations of molecular formulas would be immense. Mass measurements of atomic and small molecular ions, with uncertainties of 1-2 parts in 109, have been made by physicists for over 20 years, e.g., using a rf-mass spectrometer for the light elements (B3) and magnetic deflection instruments for the heavier elements (B4). More recently mass measurements of some lighter elements, with uncertainties in the order of 1 in 1010, have been achieved for single ions in a Penning trap. A brief overview of these achievements, and of their implications for the Avogadro constant and other fundamental quantities, has been published by DiFilippo et al. (B5). A detailed description of the meticulous experimentation, consistency checks, and error analysis required to achieve such measurements has been published by the same group (B6). Briefly, the combination of long observation times with well-understood dynamics of a single charged particle (rather than an ion cloud for which spacecharge effects lead inevitably to uncertainties at some level of accuracy and precision) form the basis for these remarkable measurements of atomic masses via ratios of cyclotron frequencies. A perspective on the implications of these measurements for various physical constants (including the Avogadro constant), and possibly for replacement of the BIPM artifactual kilogram standard by an atomic property, has been given by Pendrill (B7). Wapstra (B8) has described the history of determinations of atomic masses, with emphasis on the relationship between mass spectrometric determinations and those deduced from nuclear reaction energies. From the point of view of the mass spectrometrist conducting mass measurements on large organic ions on an essentially routine basis, with precision and accuracy currently at the level of a few parts in 106, these physics experiments (B5-B8) are of direct importance only insofar as they affect the values of fundamental constants. However, as emphasized by Dougherty et al. (B2), this may not always be true if high-field FT-ICR instruments become available. In order to minimize some of the uncertainties associated with mass corrections for sublimation and ionization energies, it was suggested (B2) that 1/720 of the mass of a single molecule of 12C60 (buckminsterfullerene) be adopted as the mass standard. One argument in favor of this proposal is that the sublimation and ionization energies of buckminsterfullerene are much smaller than those of graphite and of single carbon atoms, respectively. This would reduce to negligible values the discrepancy between the mole as defined in terms of either condensed or gaseous phase species (B3). However, Williams et al. (B9) have argued against the proposal of Dougherty et al. (B2). The adoption of the current definition of the socalled “unified” atomic mass standard was the result of a lengthy discussion involving physicists and chemists, as described by Duckworth and Nier (B10), and served to satisfy the requirements of both groups of scientists. One argument advanced (B9) in defense of the current “unified” standard lies in its experimental convenience for those engaged in measuring atomic masses of heavier elements. Most such measurements are performed using magnetic deflection instruments, by measuring mass differences between the ion to be determined and a secondary standard of mass as close as possible to that of the unknown. Typically, hydrocarbon molecular ions are used as these secondary standards in view of their almost unlimited variety. This convenience would not be possible if buckminsterfullerene were selected as 602R
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the primary standard. Derivation of the mass of an isolated 12C atom from that of buckminsterfullerene involves the atomization energy of the latter, which is of the same order of magnitude as that of graphite with a similar uncertainty. Perhaps a more telling criticism of the buckminsterfullerene proposal (B2) is a consequence of the molecular vibrations (B9). The zero-point energy of gaseous C60 is 10.2 eV (about 11 nu), but this is readily accounted for. However, excitation of vibrations implies that the mass of the molecule has a temperature dependence which is significant at the levels of precision now available in the best mass measurements (B5-B8). Even more problematic is the fact that the mass measurements are invariably made on ions, which in the case of C60+• are generally formed with a (largely unknown) distribution of internal energies and thus of masses. Whatever may be the outcome of this debate (B2, B9), the fact that it is being conducted at all reflects recent advances in precision and accuracy of mass measurements of gaseous ions. On a level more directly related to experiments conducted by chemical mass spectrometrists, Zubarev et al. (B11) have extended earlier considerations (B12) of the use of isotope-averaged masses for large biomolecules. This work (B11, B12) represents a continuation of that of Yergey et al. (B13), who concluded that the average mass, measured as the centroid of the isotopic distribution of an ionized biomolecule, is the least ambiguous of the several choices available (e.g., nominal, monoisotopic, most abundant). Later, Pomerantz and McCloskey (B14) pointed out that, because of natural variations in isotopic abundances, even theoretically calculated average masses of large molecules have a significant uncertainty which can be as large (B15) as 8 ppm for a protein when considering only the carbon content. Zubarev et al. (B11) estimate that this uncertainty would rise to 10 ppm when variations in isotopic distributions of all constituent elements are considered. They emphasize (B11) that any comparisons of experimental mass measurements derived from unresolved isotope distributions of biomolecules with theoretical values are meaningful only at the uncertainty level of 10 ppm or greater, for molecules of mass 10 000 u or greater. In addition, unless care is taken to include the entire isotope distribution in the centroid determination (with implications for both the required numbers of ions recorded, and also for thresholds used to determine mass centroids), the intrinsic shapes of isotope distributions lead to a systematic error which can be significant (B11, B12). As discussed above, precise and accurate measurements of atomic masses are intimately connected to measurement of the Avogadro constant and to the definition of the mole. For a recent discussion of these issues, see ref B16. Despite the fact that the latter is the fundamental unit of amount of substance, analytical chemists most commonly report results of quantitative analyses in terms of mass of analyte. This practice is most likely the result of habit and convenience. A proposal to conduct a program of international comparability of quantitative analyses, one objective of which is stated to be achievement of direct traceability to the mole, has been made recently (B17). For the latter purpose, reference materials of pure elements will be analyzed by ICPMS using isotope dilution techniques. While the international comparability aspects of this exercise are clear, it is not apparent from the published description (B17) just how the direct connection to the mole is to be established. Given the flood of publications on development of new mass spectrometry techniques and their applications in a wide range
of areas of interest, the role of reviews and book chapters is ever more important. Mass Spectrometry Reviews (B18) fills an essential role in this context. The Journal of Mass Spectrometry (B19), which started publication in 1995 as a continuation of Organic Mass Spectrometry but incorporating also Biological Mass Spectrometry (both of the latter have now ceased independent existence), regularly publishes special review features on timely topics. Special issues of the International Journal of Mass Spectrometry and Ion Processes over the last two years have included a volume (B20) honoring Christoph Ottinger for his pioneering contributions to fundamental studies of gaseous ions, one devoted to papers on fullerenes and other carbon and metalcarbon clusters (B21), another on secondary ion mass spectrometry (B22), a special issue on ion-molecule reactions in honor of David Smith (B23), and an honor issue (B24) commemorating the life and work of Alfred O. C. Nier, whose death due to an accident in May 1994, just before his 83rd birthday, represented a permanent loss to the mass spectrometry community to which he had contributed so much over 60 years. His career was also celebrated in a special issue (B25) of Accounts of Chemical Research. Both of these honor issues (B24, B25) cover a very wide range of subjects, in keeping with the pervasive influence of Nier’s work. The Journal of the American Society for Mass Spectrometry has also published two special issues over the same time period, one (B26) in honor of Klaus Biemann and the other (B27) in honor of Fred McLafferty. By its nature, Rapid Communications in Mass Spectrometry does not publish many review articles, but Lammert’s annual updates of the Directory of Mass Spectrometry Manufacturers and Suppliers (B28, B29) are extremely useful. In addition, this journal periodically publishes a group of papers exemplifying the work of well-known centers for mass spectrometry around the world. A new journal, European Mass Spectrometry, commenced publication in 1995 (B30). The A-pages of Analytical Chemistry continue to feature brief review articles of interest to mass spectrometrists. On-line coupling of mass spectrometers to high-resolution separation techniques was covered exhaustively in the 1994 edition of the present review, and here we draw attention only to two recent special issues of the Journal of Chromatography A, one dealing with hyphenated and coupled-column techniques (B31) and one containing papers from the 11th Montreux Symposium on liquid chromatographymass spectrometry and related techniques (B32). Recent books and monographs relevant to the present review, and not mentioned in the 1994 edition, include Laser Ionization Mass Analysis edited by Vertes, Gijbels, and Adams (B33), Applications of Mass Spectrometry to Organic Stereochemistry edited by Turecek (B34), Forensic Applications of Mass Spectrometry edited by Yinon (B35), Experimental Mass Spectrometry edited by Russell (B36), Secondary Ion Mass Spectrometry: SIMS IX edited by Benninghoven, Nihei, Shimizu, and Werner (B37), Practical Organic Mass Spectrometry: A Guide for Chemical and Biochemical Analysis by Chapman (B38), Practical Applications of Ion Trap Mass Spectrometry edited in three volumes (B39B41) by March and Todd, Mass Spectrometry in Cancer Research by Roboz (B42), Guide to Mass Spectrometry by Busch and Lehman (B42), Advances in Mass Spectrometry, Vol. 13, edited by Cornides (B44) as the proceedings of the 13th International Conference on Mass Spectrometry held in 1994 in Budapest, Hungary, Mass Spectrometry in the Biological Sciences edited by Burlingame and Carr (B45), Mass Spectrometry for Chemists and
Biochemists by Johnstone and Rose (B46), Mass Spectrometry for Biotechnology by Siuzdak (B47), Gas Chromatography and Mass Spectrometry: A Practical Guide by Kitson, Larsen and McEwen (B48), Biochemical and Biotechnological Applications of Electrospray Ionization Mass Spectrometry edited by Snyder (B49) from a symposium sponsored by the American Chemical Society, and Mass Spectrometry in the Biomolecular Sciences edited by Caprioli, Malorni and Sindona (B50). In addition to these recent titles, it is noteworthy that a new (4th) edition of Interpretation of Mass Spectra has been written by McLafferty and Turecek (B51), and that the classic 1976 text on Quadrupole Mass Spectrometry and its Applications, edited by Dawson (B52), has been reprinted by the American Institute of Physics in its AIP-AVS Classics Series (B53). The scope of a review is defined as much by what is omitted as by what is included. Thus, in the 1994 version of this review, an appreciable effort was devoted to attempts to summarize the then current status of mass spectrometers based upon trap devices, both FT-ICR and quadrupole (Paul) ion traps. Restrictions on space and available time have forced us to omit detailed discussions of these topics from the present review, although novel applications of both techniques are described in appropriate sections, below. However, it seemed important to record briefly some important advances in the techniques themselves which have been established during the last two years. The most important of these innovations seems likely to be that of ion axialization in FT-ICR, in which the radial distribution of ions in the FT-ICR cell can be reduced in a controlled manner by transferring energy from the magnetron motion (the mode generally responsible for ion loss from the cell) to the cyclotron motion (for which collisional losses of kinetic energy lead to collapse of the ion trajectories on to the cell axis). Guan et al. (B54) have written an extensive review of both the theory behind this principle and its mode of application to a wide range of experiments to provide improved signal/noise ratios, improved mass resolving power, higher mass selectivity for MS/MS, higher CID efficiency, improved efficiency of ion capture from external ionization sources, and higher efficiency of ion remeasurement techniques. As pointed out by Guan et al. (B54), full practical realization of the advantages of the FT-ICR technique has been hindered until now by the effects of the wide and generally uncontrolled radial distributions of ions in an ICR cell, but the new ion axialization procedures seem likely to overcome many of the barriers to more routine exploitation of FT-ICR in analytical and bioanalytical applications. Examples of such applications include achievement of attomole analysis of peptides and phospholipids using MALDI (B55) and a demonstration (B56) of a long-standing goal of FT-ICR (see the 1994 edition of this review) to exceed the performance of a high-resolution double-focusing instrument in molecular ion-type analysis of aromatic fractions from oil samples. While this work (B56) represents something of a breakthrough in the demonstrated applicability of FT-ICR techniques to real-world analytical problems, the authors were also commendably meticulous in delineating those areas that require futher improvement before FT-ICR can be established as the method of choice for routine molecular ion-type analyses of oil samples. Such a scientific approach, in which the remaining problems are emphasized in addition to the new achievements, should be applauded in those cases where it is to be found in the current literature. It also seems appropriate to mention two rather Analytical Chemistry, Vol. 68, No. 12, June 15, 1996
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different novel approaches to the study of interactions of trapped ions with electromagnetic radiation. Huang et al. (B57) have described an experimental approach to obtain infrared spectra of gaseous ions, using a combination of a special ICR cell incorporating ion axialization techniques plus the cavity ringdown method for obtaining absorption spectra at low fractional absorbances. Very recently, Price et al. (B58) described the exploitation of dissociation of trapped ions by black-body radiation emitted from the ICR cell walls, a subject discussed at some length in section D, in characterization of large biomolecule ions. This method is highly selective for low-energy fragmentations, which provide only limited structural information, but the authors (B58) demonstrate the use of the technique in obtaining reliable activation energies for the observed fragmentation reactions and discuss possible useful applications. The extensive review of ion optics and other design principles for ICR cells by Guan and Marshall (B59) also includes an interesting account of the historical development of ion traps of all kinds. Finally in this brief overview, we note the remarkable achievements of Smith and his collaborators (B60B63) in detection of a single macromolecular ion stored in an FT-ICR cell and characterization of its charge state. Advances have also been made over the last two years in techniques that exploit the properties of the quadrupole ion trap (Paul trap). The remarkable ability of such devices to provide high mass resolving power, principally by scanning slowly over a limited mass range, was commented on in the 1994 edition of this review. As for the FT-ICR technique, it has not been possible to provide here a properly detailed account of subsequent advances, and the following brief summary includes only some selected highlights. The achievement of high resolving power is of limited value unless some correspondingly high level of performance in mass measurement can also be achieved. Cleven et al. (B64) reported on the sometimes subtle effects of ion-ion interactions on mass shifts, which were shown (B64) to vary linearly with ion abundance at low values and to depend on the populations of other ions present in the trap as well as on the pressure of helium buffer gas which controls the ion density in the ion packet. Remarkably, Cleven et al. (B64) also demonstrated that the observed mass shifts are also compound dependent, in agreement with earlier findings of Bortolini et al. (B65). Extrapolation to zero ion abundance and to zero helium pressure were proposed as a means of improving the attainable accuracy and precision of mass measurement, but the practical applicability of such a strategy is limited. Simulation studies of the collective motions of ions in a Paul trap by Julian et al. (B66) identified the phase relationship between the rf (drive) and ac (excitation) frequencies as a significant determinant of the time of ejection of ions. A subsequent systematic experimental and simulation study due to Londry and March (B67) confirmed the findings of Julian et al. (B66) with respect to this phase relationship, which can be optimized, and further identified the noise level on the rf drive amplitude as a significant factor in determining apparent mass shifts. More recently, Cox et al. (B68) undertook a systematic investigation of the effects of space charge, categorized as the charge interactions with (a) the total number of charges in the trap, (b) the number of ions of the same m/z value as that under observation, and (c) the abundances and mass differences of neighboring ions. Effects b and c were shown (B68) to be the most important in that, as ions of a particular m/z value were isolated by stepwise elimination of neighboring ions of lower, and 604R
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more particularly, higher m/z values, the observed m/z of the ion of interest shifted to progressively lower values. Lammert et al. (B69) devised an elegant experiment in which a population of ions in a Paul trap was displaced from its central position by a fast dc pulse applied in the axial direction, and the subsequent relaxation was monitored by a laser probe which recognized when ions reappeared at the trap center via formation of photodissociation fragment ions. This technique was used (B69) to compare observed frequency components of the relaxation with those predicted by analytic theory and by computer simulations. More recent extensions (B70, B71) of these simulation and experimental studies of collective ion motion initiated by a fast dc pulse have shown that the result is to force the ions into coherent motion with characteristic secular frequencies. These observations (B70, B71) have led to a proposal (B71) for a nondestructive and massselective detection mode using the image current generated by the coherent motion, analogous to the standard detection mode used in FT-ICR by exploiting the coherent cyclotron motion. On a somewhat different aspect, further development of methods to isolate ions of a selected m/z value, while ejecting all other ions from the trap, has been reported for the stored waveform inverse Fourier transform (SWIFT) method (B72, B73), and for the filtered noise field technique (B74, B75) and other waveforms (B76). Duckworth et al. (B77) have described several other mass-selective ion storage techniques. All of these methods (B72-B77) are, of course, designed to enhance the usefulness of the ion trap in MS/MS experiments. Finally, Taylor et al. (B78) have published an admirably objective comparison of the performance of a commercial ion trap compared with that of a triple quadrupole instrument, for LC/MS analyses of in vitro metabolites of an antidepressant drug, using APCI. Some articles on subjects that are not usually associated with the present review, but which seem likely to be of interest to its audience, will now be described briefly. A review of recent developments in high-throughput stable isotope analysis has been written by Barrie et al. (B79). Chemical characterization of single aerosol particles, by combining rapid laser desorption with either time-of-flight or ion trap mass spectrometry (the latter incorporating circuitry that can levitate the macroscopic particle in the trap), is currently of considerable interest (B79-B84). Accelerator mass spectrometry is a highly specialized technique which can be used in amenable cases to quantitate rare isotope abundances as low as 1 part in 1014 using small samples. This level of performance is achieved through elimination of interfering molecular ions by high-energy collisional dissociation, avoidance of isobaric atomic interferences by discrimination via conversion to negative ions, and reduction of detector background by highenergy ion counting techniques. The best known of such applications is to 14C determinations, in which as few as 106 atoms of 14C can be determined in less than 1 mg of modern carbon more rapidly and precisely than is possible through radioactive counting techniques, as discussed in recent reviews (B85-B88). Atomic mass spectrometry, for trace determinations of metals and other elements, does not fall within the scope of the present review but recent articles on plasma mass spectrometry (B89, B90) provide useful overviews. Seubert (B91) has written a review of current practice in on-line coupling of HPLC and ICPMS, with reference to preconcentration and elimination of matrix and other spectral interferences as well as to inorganic speciation. A comparison between ICPMS and neutron activation analysis
(NAA) (B92) has concluded that each method has indisputable advantages in certain applications, but that ICPMS can replace NAA for many routine analyses. Thermal ionization mass spectrometry is currently the most accurate method for precise determination of isotope ratios in solid samples, as described in a review by Heumann et al. (B93). Resonance ionization laser spectroscopy combined with time-of-flight mass spectrometry can provide high sensitivity plus elemental and isotopic selectivity (B94), with applications ranging from studies of short-lived isotopes at on-line mass separators to a wide variety of trace analysis applications for radioactive isotopes. The use of on-line mass spectrometry for monitoring and control of plant and pilot plant operations has been discussed (B95-B97). The role of modern mass spectrometry in characterizing synthetic polymers and related materials is the subject of an excellent review by Sheng et al. (B98). The use of MALDI mass spectrometry in the analysis of dendrimer and dendrimer-linear block copolymers was shown to yield direct information on molecular weight distributions in accord with results from size exclusion chromatography (B99), while electrospray ionization has been used (B100) to provide information on structural errors in the synthesis of poly(amidoamine) dendrimers. Polyester paint resins have been analyzed using electrospray ionization together with tandem mass spectrometry (B101) and the results shown to yield detailed information on parameters such as average molecular mass, polydispersity, average frequency of branching, distributions of end groups, etc. A recent review (B102) has described the application of Curie-point pyrolysis mass spectrometry, in conjunction with artificial neural network analysis, to the systematics of bacterial classification. The use of soft ionization techniques in determinations of specific activities of tritiated compounds has been discussed (B103). An intriguing application of electrospray mass spectrometry to the study of self-assembly of complex structures around labile metal ions has been described by Williams et al. (B104). In these processes up to 20 bonds can be formed almost instantaneously to give a single product with a highly symmetrical structure such as a double or triple helix (B104). As a final example of this highly idiosyncratic list of applications off the main track of the present review, Limero’s description of mass spectrometers aboard U.S. spacecraft (B105) is of general interest. INNOVATIVE TECHNIQUES AND INSTRUMENTATION The current breadth of impact of mass spectrometry in the life and physical sciences is testament not only to the fundamental attributes of the technique but to the continuing ingenuity displayed by those who develop new instumentation and (equally importantly) those who apply mass spectrometry, often in areas of interdisciplinary research. As with previous reviews in this series, it has been necessary to apply considerable selectivity in the choice of topics covered in this section. The principal criterion has been that the innovation has found widespread application (or shows promise of doing so). Furthermore, the perennial inclusion of this section in the biennial review allows us to emphasize topics which perhaps received short shrift on previous occasions. Nevertheless, it is readily acknowledged that the selection remains subjective (reflecting the prejudices of the reviewers) and the omission of a particular topic is by no means intended to imply a lack of innovation or significance.
Membrane Inlet Mass Spectrometry. The on-line combination of mass spectrometers with separatory techniques, most often chromatographies of various kinds but more recently also capillary electrophoresis, has led to its unique role in qualitative and quantitative analyses of trace components in complex mixtures. The principal disadvantage of the chromatography/MS combinations is their reliance on separation in time of the components of a mixture. Therefore, such techniques are inherently limited in their turnaround time per analysis and thus in their applicability to continuous process monitoring, for example. Accordingly, there has been a revival of interest in developing analytical methods based on direct introduction mass spectrometry. Obviously the complexity of the mixtures amenable to such techniques is limited, even when combined with chemometric methods such as principal component analysis to assist with data interpretation or with tandem mass spectrometry to increase the information content of the mass spectrometric data obtained. Nonetheless it has become apparent that there is an important niche for direct introduction mass spectrometry in continuous monitoring of process streams or of wastewater, or in on-site environmental testing in locations where practical difficulties in operating, e.g., a GC/MS system, would be prohibitive. The analysis of volatile organic compounds present at trace levels in liquids (particularly water) normally requires extraction and preconcentration steps, followed by, e.g., GC/MS analysis. If the liquid sample is to be presented directly to the mass spectrometer, it is necessary to find a means of selectively sampling the target analytes while excluding the bulk of the solvent. Over the past few years a considerable effort has been devoted to optimizing membrane inlet mass spectrometry (MIMS) for this purpose. Some recent reviews by Cooks and his collaborators (C1-C5) have provided excellent coverage of this work through 1995. The present brief account draws heavily on these reviews and also includes some more recent developments. A recent review (C6) of mass spectrometry applied to on-site environmental analysis and to process monitoring also includes a useful account of MIMS in the context of competing methodologies. The introduction section of a recent paper on application of MIMS to analysis of volatile organic compounds in seawater (C7) provides an excellent brief introduction to the subject. Successful analysis of water samples by MIMS requires above all that the water be excluded from the ion source, and this implies a requirement for hydrophobic membranes. Silicone rubber membranes have been commonly used, but the price to be paid is a restriction on the molecular masses ( 70 the loss of single Si atoms is the preferred fragmentation channel, and indeed bulk silicon evaporates off single atoms when heated. These trends can be understood (D53) in terms of the thermodynamics of the dissociations. Germanium lies immediately below silicon in the periodic table, and the IC behavior of Gen+ clusters shows intriguing differences from that of their Sin+ counterparts. For clusters with n < 35 or so the relative mobilities decrease sharply with increasing size and then level off at a constant value for n > 55 or so (D53). Annealing experiments resulted in a change in the ATD spectrum only for n ) 40, for which a low-mobility isomer was formed. Together with measurements of dissociation energy onsets (D60), with Ge10 being the only neutral product formed, it was possible (D53) to acquire some understanding of the trends in the properties of the Gen+ clusters. Metal clusters are expected to adopt roughly spherical shapes, since they are not subject to the directional restrictions imposed by covalent bonds as in the case of the semiconductor elements. This appears to be essentially true, but shapes of aluminum cluster ions, for example, as indicated by IC determinations of mobilities determined by Jarrold and Bower (D61, D62), are modulated by quantum size effects. The electronic shell model (D63) accounts for many of the properties of clusters of free-electron metals by assuming that the delocalized valence electrons move in a uniformly positively charged background (the “jellium” model), so that the electronic energy levels are quantized by angular momentum restrictions in a manner analogous to that applicable to the well-known case for atoms. There are thus electron shells which can be closed, with concomitant discontinuities in mobilities as measured by the IC technique. In some cases isomeric structures exhibiting annealing behaviour were observed (D61, D62). Carbon cluster ions exhibit behavior much more complex than that of their Si or Ge analogues, in accord with the unique richness
of organic chemistry. Bowers and his collaborators have expended considerable effort in applying the IC technique to the problem of isomeric structures of both positive and negative Cn cluster ions. Much of this work has been summarized in two recent review articles (D50, D52), and it will be possible here to outline only the highlights of this work (D64-D73) which has greatly clarified the mechanisms of the growth of carbon clusters and of the formation of fullerene structures. A combination of experiment (ATDs) and theory (molecular orbital calculations of isomer structures, and a Monte Carlo approach to calculation of theoretical mobilities for these calculated structures) permitted assignment of structures to the various families of isomers observed in the IC spectra for carbon cluster cations formed by laser desorption (D71). For Cn+ species, linear structures exist up to n ) 10. Monocyclic ring structures first appear at n ) 7 and persist up to n ) 40, while bicyclic ring structures first appear at n ) 21 and persist above n ) 40. Tricyclic rings appear at n ) 30, and tetracyclic rings at n ) 40, with well-defined theoretical structures (“propellers” and “linear” arrangements of rings) providing excellent agreement with experiment (D71). A lowabundance family of three-dimensional structures, as yet undetermined, was observed at n ) 29 and above. The first fullerene was observed at n ) 30, and this family was observed to dominate above n ) 50. The growth pattern of Cn+ clusters with increasing n was shown (D71) to follow the sequence linear, monocyclic rings, polycyclic rings, fullerenes. No PAH-like (graphitic) isomers were observed as intermediates to formation of fullerenes (D71), and indeed annealing experiments (D66, D67) have suggested a mechanism for fullerene formation whereby essentially planar polycyclic ring structures with n > 30 can be activated to form fullerene structures with simultaneous expulsion of small neutral carbon clusters. This represents a remarkable amount of information derived from measurements of an apparently chemically uninteresting parameter (gaseous ion mobilities) and illustrates yet again the importance of applying the best available theoretical models to the interpretation of experimental data. Additional work (D73) on the corresponding carbon cluster anions has demonstrated that same structure families are observed, although the stability ranges are quite different. The same growth pattern is also observed, but the fullerenes do not dominate in the same way at higher n values. For example, for even values of n near 60, fullerenes comprise more than 95% of the cation isomer distribution, but the corresponding figure for the anions is less than 20% with planar ring structures dominating. More recently, Jarrold et al. have extended these carbon cluster experiments to MCn+ clusters, where M is a transition metal. For example, for M ) La (D74) the structures included three families of metal-containing carbon rings, metal-containing graphitic sheets and, metallofullerenes (both endohedral and nonendohedral). However for M ) Pd no metallofullerene structures could be found, and when annealed the Pd atom was lost to form the fullerene cation (D75). In somewhat related work, Bowers et al. (D76) have investigated the cations of the “met-cars”, or metallocarbon composites M8C12 discovered by Castleman et al. (D77, D78), and showed that the dodecahedron structure originally proposed (D77, D78) gave the best fit to the IC data. In the first experiments conducted using their tandem hybrid IC instrument, Kemper and Bowers (D57) made the astonishing discovery that it is possible to separate electronic configurations of Co+ ions via their gas-phase mobilities in helium. Extensive Analytical Chemistry, Vol. 68, No. 12, June 15, 1996
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investigations of this phenomenon (D79, D80) confirmed that all first-row transition metal ions show this same behavior and that states with a 3dn configuration had lower mobilities than those with a 3dn-1 4s1 configuration. The 3dn ions had mobilities that precisely matched those predicted by Langevin theory, indicating that these ions undergo capture collisions (ion-induced dipole orbiting complexes). The greater mobilities of the faster 3dn-1 4s1 ions were rationalized on the basis of repulsive interactions between the electron in the 4s orbital of the metal with the complete 1s2 shell of the helium atom, which prevented capture collisions from occurring. This separation of electronic configurations has been exploited in studies of state-selective reactivities, for example with C3H8 (D81) and CH3I (D82). More recently (D83-D85) Bowers et al. have fitted a MALDI source to their IC instrument and investigated sodiated PEG oligomers and crown ethers complexed with alkali metal ions, they have also reported some preliminary investigations on peptide ions. Jarrold et al. (D86) have added an electrospray ion source to their double-quadrupole IC instrument and have investigated conformations of unsolvated bovine cytochrome c ions by the IC technique. For the +7 charge state three distinct peaks plus a broad low-intensity shoulder at short drift times were observed in the ATD spectrum. With increase of charge state the peaks at shorter drift times progressively disappeared, and the ATDs for the +9 to +20 charge states consisted of a single peak correlating with the longest drift time observed in the +7 ATD, for which the drift times (and the collision cross sections deduced from them) increased slowly with charge state (D86). Model calculations showed that the predicted mobilities for the native (folded) and for a near-linear conformation are too small and too large, respectively, and that the dominant peak in the ATD spectra must correspond to a partially folded conformer. The best fit was obtained for a conformer of the type in which the secondary structure is retained but the tertiary structure is lost (D86). Another measurable quantity, which is interpretable to give values of the cross sections for the elastic collisions which dominate the IC experiments, is the translational energy loss of the ions as they traverse the drift cell. Experiments of this kind have been reported by Covey and Douglas (D87), who used a conventional triple-quadrupole tandem instrument and measured translational energies from stopping curves observed as the offset potential of Q3 was varied. Cross sections for several protein ions in various charge states, in collision with argon, were derived from the energy loss data using a simple collision model in a Monte Carlo strategy (D87), but later Douglas (D88) modeled the phenomenon as the aerodynamic drag on a projectile at high Knudsen number (ratio of the mean free path of the collision gas to the diameter of the projectile). The absolute values of cross sections so derived (D88) were some 80% of those from the discrete collision model (D87) and are in reasonable agreement with those derived from measurements of mobilities (D86). The increase of cross section with ion charge was greater than that predicted by a simple Langevin model, and it was suggested (D87) that this may reflect more extended structures for the higher charge states of these proteins. For cytochrome c, the cross sections for ions formed by electrospraying 1:1 water-acetonitrile mixtures were significantly larger than those obtained for ions electrosprayed from a 1:9 water-acetonitrile mixture (D87), suggesting that the gaseous ions retained some memory of their conformation in solution prior to the electrospray process. More 618R
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recently, Douglas (D89) has applied these same techniques to a study of gas-phase horse heart myoglobin ions. There had been a previous suggestion (D90) that gaseous myoglobin ions may have a more compact structure than the corresponding apomyoglobin (formed by loss of the heme prosthetic group bound to the protein moiety by van der Waals interactions, hydrogen bonds, and coordination of the heme iron to ligating side chains). Cross sections measured (D89) as a function of charge state confirmed this previous suggestion in a more quantitative way. Some information on the mechanism of heme group expulsion, following collisional activation in the skimmer region of the electrospray source, was obtained from results using moderate values of the orifice-skimmer potential difference such that only some of the myoglobin ions were converted to their apomyoglobin counterparts. These activated but undissociated myoglobin ions had cross sections very similar to those of the apomyoglobin ions, suggesting (D89) that the myoglobin ions upon activation first unfold to give undissociated species which can survive for the 1 ms or so required for passage to the detector, and that it is these unfolded ions which expel the heme group if sufficiently activated. Cox et al. (D91), in related experiments using a triple-quadrupole instrument, showed that the bimodal charge-state distribution observed in the electrospray mass spectra (Q3 scan) of horse heart myoglobin could be dramatically changed by variations in the pressure of argon added to the central (rf-only) quadrupole. A high charge-state distribution centered at +20 predominated at low pressures, and the other centered at 10+ at high pressures. It was confirmed (D91) that these effects were not due to collisioninduced dissociation, but to larger elastic collision cross sections for the conformers giving rise to the higher charge-state distribution. This resulted in translational energy losses sufficiently large that the high charge-state ions were not extracted efficiently into Q3. These observations were interpreted (D91) in terms of a more open conformation corresponding to larger cross sections than the more folded conformations and providing access to a larger number of basic sites for protonation. Gaseous Ion Thermochemistry. The correlation and critical evaluation of all the disparate information relating to the thermochemistry of gaseous ions is an enormous and complicated task, exemplified by the ongoing efforts of Lias et al. (D92-D94). Here we discuss only a few relatively clearcut advances which have been made in the last few years. The establishment of a quantitative gas-phase proton affinity (PA) scale has been a major undertaking since the late 1960s. Relative values of PA have been obtained via measurements of equilibrium constants for proton-transfer reactions using ICR, highpressure mass spectrometry, or flow methods, and remarkable progress has been made in improving both accuracy and precision (around 2 kJ mol-1) of these values of PA differences. In order to establish an absolute scale it is necessary (D92-D94) to fix the scale to several “known” proton affinities generally determined from appearance energy measurements. For example, the PA of isobutene was chosen (D92) as one of these primary standards, based upon measurements of the appearance energy (and thus enthalpy of formation) of the tert-butyl cation from a variety of neutral precursors with known heats of formation. Together with known values of the heats of formation of gaseous H+ and of isobutene neutral, the PA of isobutene could in principle be determined in an absolute sense.
Most of the early PA scales were based upon single-temperature measurements of equilibrium constants, i.e., measurements of changes in free energy from which enthalpy changes had to be derived using estimated entropy data. Meot-Ner and Sieck (D95) in 1991 published the first set of temperature dependences of proton-transfer equilibrium constants which linked a number of bases that could be regarded as primary standards, based upon reliable appearance energy measurements and associated subsidiary data. In this way each of the enthalpy and entropy changes was measured directly. One of the most notable findings of this work (D95) was that the PA difference between isobutene and ammonia was larger by >26 kJ mol-1 than previously assigned (D92). This greater difference was confirmed in an independent study by Szulejko and McMahon (D96). These new experiments seemed to demand a higher PA value for NH3, and a consequent readjustment of a large part of the upper region (most basic compounds) of the gas-phase PA scale. Interestingly, this interpretation (D95, D96) of the larger PA difference was challenged practically simultaneously on the basis of both high-level (G2 level) molecular orbital calculations of Smith and Radom (D97) and experimental measurements due to Szulejko and McMahon (D98) of temperature dependences of proton-transfer equilibrium constants for approximately 80 base pairs, covering the basicity range from nitrogen to tert-butylamine. The conclusions drawn from these theoretical and experimental investigations (D97, D98) were in gratifying agreement, with a mean difference between theory and experiment of 2.4 kJ mol-1 for the 15 cases where comparisons were possible and a maximum difference of 7.0 kJ mol-1. The most important finding was that it is the PA of isobutene which is the primary standard requiring major revision, downwards by 10-20 kJ mol-1 (D97) or by approximately 17 kJ mol-1 (D98). The PA of ammonia was lowered by only some 2 kJ mol-1 as a result of these revisions (D97, D98). The discrepancy for isobutene called for reexamination of the appearance energy determinations for the tertbutyl cation from dissociative photionization of tert-butyl halides (D99), on which the previously accepted PA value had been based (D92). A PIPECO measurement of the dissociative photoionization onset of tert-butyl iodide by Keister et al. (D100) used cooling in a supersonic molecular beam to reduce to negligible values the thermal shift discussed for such experiments by Chupka (D101). The value for the PA of isobutene at 298 K thus derived by Keister et al. is 802 ( 3.6 kJ mol-1, in agreement to within 1 kJ mol-1 with the new theoretical (D97) and experimental (D98) values. It is of interest to note that the thermal shift effect is also minimized if the ionization energy of the neutral free radical (tertbutyl in this case) is determined even at ambient temperatures, as discussed by Chupka (D101) for photoionization onset measurements. In fact, as early as 1970 Lossing and Semeluk (D102) determined the ionization energy of the tert-butyl radical using monochromatic electrons, for which the thermal effect is again predicted to be small (D103), and this value combined with modern values for subsidiary data yields (D98) a PA for isobutene of 793 ( 5 kJ mol-1, in reasonable agreement with the foregoing discussion. Very recently Traeger (D104) has repeated his measurements (D99) of dissociative photoionization onsets for tert-butyl cations from several neutral precursors and obtained results which yield PA values for isobutene in satisfactory agreement with the other recent determinations (D97, D98, D100).
It is important to realize that the PA for isobutene has a significance far beyond its own intrinsic importance, in view of its adoption as one of the fixed standard points on the PA scale established by Lias et al. (D92). As a result of the consensus to lower this PA value by 17-18 kJ mol-1, the self-consistency of the entire PA scale is now greatly improved (D97, D98). This is another example where the best modern techniques of theory and experiment give results which converge, thereby increasing confidence in both. Recently, Smith and Radom (D105) have improved their G2 computational method to give PA values within 10 kJ mol-1 of the best experimental data, but at significantly lower computational cost. The critical evaluation of the energetics of negative gaseous ions has in the past received less attention than for positive ions, but thanks to the efforts of Bartmess (D94) this situation is changing for the better. Bartmess has also made a fundamental contribution to the entire field of gaseous ion thermochemistry (D106) by his critical evaluation of the statistical thermodynamics of the free electron. These considerations are of direct importance for energetics of formation of radical cations and anions, but also have implications for PA values via the thermochemical properties of the free proton which are derived from those of the hydrogen atom and of the electron. The situation is complicated, but has two principal components (D106). The first of these concerns thermodynamic standardstate conventions, which arbitrarily assign the enthalpy and Gibbs free energy of formation of each and every element to be zero at all temperatures (D107). For the free gaseous electron, the socalled “electron convention” (EC) regards the electron as an element in this sense and evaluates the heat capacity and entropy as that of a classical ideal gas with statistical degeneracy of 2 to account for the spin quantum number. In contrast the “ion convention” (IC) assigns zero enthalpy of formation to the electron at all temperatures, as in the EC, but in addition assigns a zero value to the integrated heat capacity (i.e., the enthalpy change from zero kelvin to any finite temperature). Unless one can admit negative values for heat capacity to balance out positive values in some different temperature range, this convention would seem to imply that the heat capacity of the electron is being assigned zero values at all temperatures, and therefore so is the entropy. Both the EC and IC have been used in important thermochemical compilations, though the most recent tables from NIST (D92D94) have used the IC consistently. Thermodynamic standard states are conveniences (D107) to facilitate the tabulation of absolute values for enthalpies and free energies of formation for individual substances. Calculation of enthalpy and free energy changes for balanced chemical reactions are not affected by the convention adopted for the elements (including the electron if included as such), since the contributions from the elements must cancel for such a process as long as a single convention is applied consistently. However, Bartmess (D106) points out that, for gasphase reactions involving the electron as a reactant forming a product anion, this cancellation does not occur and a full thermodynamic treatment of the electron is necessary. The second problem relates to the integrated heat capacity and entropy of the electron at nonzero temperatures. The IC defines both of these to be zero at all temperatures, an unrealistic assumption since it must require energy to raise the temperature of an assembly of particles. However, as pointed out by Bartmess (D106), this assumption of the IC is no more irrational than Analytical Chemistry, Vol. 68, No. 12, June 15, 1996
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defining the enthalpy of formation of an element to be zero at all temperatures and simultaneously defining the integrated heat capacity to be nonzero (D107). The EC, on the other hand, treats the free electron like any other element without any additional arbitrary assignments. However, the numerical evaluation of heat capacities and entropies of the electron within the EC has in the past been done using classical Bolzmann statistical mechanics, e.g., the Sackur-Tetrode equation for the translational entropy (D108). However, the Sackur-Tetrode equation applies to boltzon particles, which are (assumed) distinguishable particles with no exchange-symmetry restrictions on the many-particle wave functions, and thus no restrictions on multiple occupancy of singleparticle states (D109). Fundamental particles such as the electron, proton, and atomic nuclei, on the other hand, are truly indistinguishable particles and must be classed either as fermions (Fermi-Dirac statistics) or bosons (Bose-Einstein statistics) (D109), and state occupancy restrictions do apply. The reason why classical Boltzmann statistics usually work so well is that, for gases at reasonably high temperatures, the number of quantum states available for occupancy is much larger than the number of particles so that the symmetry restrictions are of negligible consequence for thermodynamic functions well above absolute zero (D109). The electron is a fermion, and due to its low mass, the density of translational states is correspondingly low so that the deviations of predictions using Fermi-Dirac statistics from those using Bolzmann statistics occur at unusually high temperatures. Bartmess (D106) has evaluated the quantitative consequences of this discrepancy. Correction of enthalpies and free energies of formation of ions for such quantum statistical effects within the EC would make these thermochemical quantities more negative by 3.086 kJ mol-1 for cations, while those of anions would increase by the same amount. In the case of the more recent compilations from NIST (D92-D94) which adopted the IC, adoption of the EC with quantum corrections would increase enthalpies of formation of cations by 3.110 kJ mol-1 while those of anions would decrease by this amount. Any changes in convention will not alter enthalpies and free energies of reaction except for cases in which the numbers of gaseous reactants and products are different (so that the corrections to translational entropies do not cancel). Particularly important in this regard are reactions in which the free electron is a reactant or product (D106). As mentioned above there are also consequences for the proton, which is also a fermion. Due to its much larger mass compared with the electron, the Boltzmann evaluations of entropy and integrated heat capacity of the free proton at room temperature are not significantly different from those calculated using Fermi-Dirac statistics. However, in both the IC and EC the proton is not regarded as an element, and evaluation of the consequences of the two conventions and of quantum statistical corrections includes the effects for the electron (via the reaction in which a proton is formed from a hydrogen atom). Bartmess showed (D106) that adoption of the EC with quantum corrections would raise the enthalpy of formation of the proton by 3.104 kJ mol-1 relative to that in the IC (D92-D94), while free energies of formation would be decreased by 3.690 kJ mol-1, with consequent (constant) changes to PA values. It may appear that these considerations (D106) are of little practical consequence, in view of the small magnitudes of the corrections. However, as noted above, there are cases where the quantitative consequences are nontrivial. Moreover, in view of 620R
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the fundamental role played by ion energetics in theoretical mass spectrometry, it seems desirable if not essential to place the conventions on a self-consistent basis and consistent also with modern physical science (D109). The discussion thus far has involved the highly accurate and precise thermochemical data obtainable from equilibrium measurements by high-pressure mass spectrometry, or by ICR or flowing afterglow/SIFT techniques. Not all ions of interest are readily amenable to investigation by these techniques, however. For this and other reasons the kinetic method was introduced by Cooks and Kruger in 1977 (D110). A definitive review of the experimental procedures, theoretical underpinning, and data obtained up to early 1994, has been published by Cooks et al. (D111) so that only a brief outline is given here. In essence the kinetic method explores the relative affinities of two moieties A and B for a charged species c, by investigating the fragmentations of the AcB species into (Ac + B) or into (A + cB). The charged species c can be a proton, an electron, a metal ion, or any (generally small) charged species. The relative intensities of the competing fragmentation channels are then interpreted in terms of the relative affinities of A and B for c (A and B are generally neutral molecules, but experiments have been reported where one or more of them is a neutral free radical or an ion). An extensive discussion of the theoretical basis for the method has been given (D111). The general question of the relationship of kinetic parameters to the thermodynamic properties of the same reaction is problematic. In particular, the question as to whether the relationship should involve changes in only energy and enthalpy, or alternatively in free energy (incorporating entropy), is frequently encountered. Fenselau et al. (D112-D114) explicitly tackled the question of entropy effects through judicious choices of the competing species A and B, which were amino acids and small peptides in these experiments. In general (D111) the kinetic method appears to provide useful data for cases where the AcB reactant species are weakly bound, e.g., proton-bound dimers which yield relative PA values. A recent extension of the kinetic method to determine the gas-phase basicities of protonated peptides, i.e., the energetics associated with acquisition of a second proton, has been described by Kaltashov et al. (D115). Other recent applications include measurements of affinities of (A,B) ) substituted pyridines for c ) carbonylisocyanate cation (D116), SiCl3+ and SiCl+ (D117), and SiF3+ (D118). Very recently (D119) the kinetic method was used to measure the gas-phase basicity of the zwitterionic compound betaine (CH3)3N+CH2CO2-, apparently the first such measurement on a zwitterionic compound. Substitution of a hydrogen atom on acetate anion by a trimethylammonium group resulted (D119) in a decrease in the basicity from 1458 to 975 kJ mol-1, an enormous substituent effect consistent with the Coulombic interactions in the zwitterion. (The basicity of the anion of N,N-dimethylglycine is 1410 kJ mol-1, still 435 kJ mol-1 larger than for the betaine zwitterion). The kinetic method has also been applied to other species A, B such as nucleosides (D120, D121), whose basicities would be difficult to determine by other techniques. A different approach to experimental thermochemical determinations of what would formerly have been regarded as exotic ions in this context has been described by Kebarle and collaborators (D122-D126). These experiments form ions by electrospray ionization at atmospheric pressure, transfer the ions to a chamber maintained at a pressure of a few Torr and to which neutral
reactants can be added at known partial pressures, and analyze the resulting equilibrium mixtures by careful sampling into a mass spectrometer. The systems studied thus far include hydration equilibria of singly and doubly protonated alkyldiamines, protonated alkylamines, protonated glycine oligomers and other small peptides incorporating lysine and tyrosine, anions of some oxo acids, and metal ions ligated with water or acetone. Two papers on hydration equilibria of phosphate anions (D125, D126) investigated orthophosphate anion (HO)2PO2-, diphosphate dianion (HO)2POP(OH)22-, ribose 5-phosphate, adenosine 5′phosphate, and adenosine 5′-diphosphate. Interconversion of metaphosphate PO3- and its hydrates with the orthophosphate counterparts was not observed (D125), despite expectations that this equilibrium should have been established. Use of the apparatus in a different mode (D127) permitted collision-induced dissociation threshold measurements for the dehydration of orthophosphate to metaphosphate, giving a high dissociation barrier of over 220 kJ mol-1 accounting for failure to establish the metaphosphate-orthophosphate equilibria under the conditions of the experiment (D125). In the case of the sugar phosphate and nucleoside anions (D126), unusually low hydration exoergicities indicated the presence of intramolecular hydrogen bonding. This new technique (D122-D126) seems likely to open up many new classes of gaseous ions to thermochemical determinations via measurements of equilibrium constants. Finally, it is appropriate to mention the empirical approaches to estimation of thermochemical quantities. These are described in the 1988 critical evaluation of thermochemical data from NIST (D92). More recent advances in this approach have included the work of Holmes and Lossing (D128), who demonstrated a remarkable simple empirical realationship between the ionization energy (IE) of a homologous series of organic compounds and n, the number of atoms in the molecule, viz., IE ∝ 1/n. The only exception investigated consisted of the unsubstituted cycloalkanes, for which the IE values fell in the range 9.80-9.83 eV (average uncertainty (0.05 eV) for cyclopentane to cyclooctane (D128). Other advances in this area include the work of Luo and Benson (D129) on derivation of a simple parameter which measures valence-state electronegativity and appears to be remarkably successful in correlating and predicting several thermochemical quantities. This work has been extended by Luo and Pacey (D130) and by Luo and Holmes (D131) to general schemes for predicting homolytic bond dissociation energies for organic compounds, quantities important in thermochemical cycles used to calculate energetics of gaseous ions. This approach has been extended by Luo and Holmes to prediction schemes for C-X bond dissociation energies in unsaturated compounds (D132, D133), for stabilization energies of polyenyl radicals (D134) and in vinyl and phenyl compounds (D135), and to heats of formation of multisubstituted positive organic ions (D136). Collisional Activation and Collision-Induced Dissociation. The title of this section is intended to reflect the common view of the overall process of collision-induced dissociation (CID), in the sense of the term as used in the context of tandem mass spectrometry, as two well-separated steps. According to the twostep mechanism hypothesis, the collisional activation (CA) step is well separated in time from the subsequent unimolecular dissociation of the activated ion. The second step involves fragmentation kinetics of activated ions and is well developed both experimentally (see Ion Dissociation Dynamics, above) and
theoretically [see the excellent review by Lorquet (D137) of modern aspects of statistical theories]. The CA step has been less well studied and understood, as emphasized by Shukla and Futrell in two recent reviews (D138, D139) which draw heavily on their own elegant studies of energy and angular distributions of CID fragment ions. A recent example of the detailed mechanistic studies enabled by their unique crossed-beam apparatus is provided by the investigation of CID of CS2+• ions (D140, D141), in which the competition between impulsive collisions (vibrational excitation) and electronic excitation, to form CS+ in competition with S+, was shown to vary with collision energy. In a more empirical vein, Vachet et al. (D142) have demonstrated an unexpectedly high degree of correlation between the loss of translational energy in the CA step for peptide ions at kiloelectronvolt energies and the type of fragment ion formed in the overall CID process. The greatest energy losses were observed for formation of a ions, followed by b and then y fragment ions (D142). A different approach to investigation of the CID process is to study the light emitted in such events. The first experiments of this kind due to Holmes et al. (D143, D144) analyzed the wavelength dependence of the emitted intensity by using a series of cutoff filters. Subsequent experiments (D145-D147) have used a scanning monochromator for greater spectral resolution. Detailed discussions of the observations are given in the original work (D143-D147), but it is notable that light emitted from kiloelectronvolt collisions of polyatomic ions with a variety of collision gases is dominated by that from atomic and diatomic fragments. The implications of this observation for the CA process itself remain to be worked out. Emissions from larger molecular fragments may be present at low intensity, and the sensitivity of the experiment should be considerably enhanced through installation of a CCD detector (D147). Somewhat similar experiments have been reported by Wiedmann and Wesdemiotis (D148), who used cutoff filters to examine photon emissions from CA of a variety of ions produced by FAB ionization. The emissions from some monatomic metal ion projectiles varied considerably as to whether the ion itself or its neutral counterpart (formed in electron capture collisions) was the dominant emitter. Cs+ and W+ are examples of the former, while use of Na+ projectiles resulted in almost all the emission corresponding to the well-known D-lines of sodium atoms (D148). The intensity of emitted light, normalized to the projectile ion current, invariably increased with size in ion series including MnIn-1+ (M ) Na, Cs) and [Ala]nH+ (n ) 1, 3, 5). An attempt to correlate CA emission spectra with functional groups in a set of organic molecular radical cations (D148) resulted in no definite trends, a result possibly related to the findings of Holmes and Mayer (D147) that the majority of the emitted photons arise from monatomic and diatomic fragments. Structures of Neutral Fragments. Interpretation of mass spectral fragmentation patterns in terms of structures of the sample molecules requires an understanding of the fragmentation mechanisms involved. Some early approaches to determining the nature of neutral products of ion-molecule reactions exploited the properties of either a time-of-flight analyzer (D149, D150) or of an ICR spectrometer (D151, D152). A specially designed dual EI ion source allowed Beck and Osberghaus (D153) to produce what appear to be the first EI spectra of neutral fragments in 1960, and this source design was adopted and modified by others [see Analytical Chemistry, Vol. 68, No. 12, June 15, 1996
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Melton (D154) for a review of this early work]. However, the subsequent design of Reeher et al. (D155), in which the intensity of the primary electron beam responsible for producing the original fragments was modulated at a well-defined frequency, permitting lock-in detection of the mass spectra of the neutral fragments which drifted into the secondary ionization chamber, represented a significant advance. The EI mass spectra of the neutral fragments could thus be recorded free of interferences from the dc signals from the much more abundant undissociated sample molecules, and their appearance energies could also be determined (D155). A more recent advance was based on the discovery that fast (keV energy range) neutral species could be subjected to dissociative ionization via collisions with thermal target gases. This phenomenon of collision-induced dissociative ionization (CIDI) has been exploited in the body of work conveniently labeled neutralization-reionization mass spectrometry (NRMS), in which a fast beam of mass-selected ions is partially converted to the corresponding neutrals through interactions with judiciously selected collision gases, residual ions are removed by electrical deflection, and the fast neutrals then subjected to CIDI in a second collision cell. The NRMS technique has been used to investigate exotic neutral species, which are difficult or impossible to prepare in any other way, and is discussed in section C. The related experiment, in which the fast neutrals are prepared as neutral fragments of spontaneous dissociation or of CID, has been studied to an appreciably lesser extent (D156-D160) until recently. This early work (D156-D160) was concerned mainly with small precursor ions, generally produced by EI or CI. However, more recently Wesdemiotis et al. have published a series of investigations (D161-D167) in which structures of neutral fragments from dissociations of much larger precursor ions have been investigated by CIDI. Many of these have involved sample molecules of biological significance, and this work up to early 1994 has been conveniently reviewed (D168). Standard fast beams of neutrals can be obtained by CID of the proton-bound dimers, thus permitting acquisition of standard spectra in suitable cases. Fragmentations of singly protonated peptides formed by FAB ionization were shown (D161) to proceed by elimination of the neutral moiety as a single unit, rather than as a series of smaller fragments. The N-terminal acylium (b) and immonium (a) ions have complementary neutral fragments corresponding to intact peptides. The largest y fragment (elimination of only one amino acid residue from the N-terminus) has an aziridinone complementary neutral fragment. Other smaller y fragments are accompanied by more stable diketopiperazine neutrals with sixmembered ring structures. In this same study it was also shown by MS/MS/MS techniques that b fragment ions are not diketopiperazines, but rather are either linear structures or protonated oxazolones (D161). Structures of the neutral fragments were shown (D162) to be indicative of the sequence of isomeric dipeptides, e.g., Ala-Gly vs Gly-Ala, and this work was later extended to larger peptides (D163). The neutral products formed by charge remote fragmentations of ions formed from fatty acids, both (M - H)- and (M - H + 2Li)+ species, were shown by CIDI (D164) to be 1-alkenes, consistent with the mechanism involving 1,4 elimination of H2 originally proposed by Jensen et al. (D169) for this class of ions. Metalated linear polyglycols (M + X)+ (X ) Li, Na, K) also preferentially fragment via 1,4 elimination of H2, but their cyclic counterparts (crown ethers) 622R
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preferentially produce distonic radical cations via losses of saturated radicals (D165). Singly and multiply charged C60 fullerene cations are well-known to fragment by losses of neutrals C2n, and it was shown by CIDI (D166) that these are intact C2n clusters rather than n consecutive losses of C2 units. Differentiation of Ile from Leu via CIDI of neutral fragments was shown (D167) to be possible provided that these residues are present as C-termini of the precursor ions. The importance of these CIDI studies of neutral fragments for the deeper understanding of the dissociation mechanisms of organic ions, thus leading to increased confidence in the spectral interpretation of unknown samples, is apparent. As emphasized by Cordero and Wesdemiotis (D168), however, the CIDI phenomenon appears to be a “hard” ionization, since molecular ions are frequently unobservable. Chemical ionization of these neutral fragments would require deceleration of the beams to near-thermal energies, and this has been achieved by Orlando et al. (D170, D171). A full realization of the combined approach has yet to be described, however. Structures of Unusual Small Ions and Neutrals. The NRMS technique has yielded otherwise inaccessible information on unusual neutral species, particularly when deployed in conjunction with high-level molecular orbital calculations of energy surfaces. A number of excellent reviews (D158-D160, D172, D173) have described the principles and applications of the technique, covering the literature up to about mid-1994. Turecek (D174) has also written an admirable review of mass spectrometric investigations of transient neutral species, in which NRMS is set in the perspective of other approaches such as flash pyrolysis. Accordingly, only some more recent work will be mentioned here. Harnish and Holmes (D175) undertook a systematic investigation of the effects of translational energy and internal energy of the fast neutrals on the efficiency of CIDI, the second step of the NRMS experiment. This work complemented an earlier study (D176) of the parameters affecting the efficiency of the electron transfer step which produces the fast neutrals. The CIDI study (D175) of C2H4 and CO, prepared with varying degrees of internal excitation by preparing them by ion dissociations whose thermochemistry is well understood, showed that the CIDI efficiency for collisions with oxygen increased with increasing translational energy and decreased with increasing internal energy. The latter effect was thought (D175) to correspond to increasing competition from homolytic CID of the neutral. Some innovative developments of the NRMS technique have been made by Turecek, including survivor ion mass spectrometry (D177-D179) in which those ions which survive the dual NRMS process are useful in isomer differentiation. Another development is that of variable-time NRMS (D180, D181) in which the delay between neutralization and reionization is varied to yield kinetic information on the fragmentation reactions of the fast neutrals and can be used to differentiate between the dissociations of the neutral and those of its ionized counterpart. Turecek recently has exploited these techniques, in combination with high-level molecular orbital calculations, in studies of several transient neutrals including hypervalent (D182, D183) and other (D184, D185) radicals. Terlouw has also been prolific in NRMS, particularly in exploitation of the technique to characterize exotic neutral species formed by electron transfer to the corresponding radical cations. Since publication of the NRMS reviews in 1994 (D172, D173) the main
focus of Terlouw et al. has been on carbenes, including diaminocarbene (D186), dihydroxycarbene (D187), hydroxyaminocarbene, and (N-methylamino)hydroxycarbene (D188), again combining experiment with molecular orbital calculations. A similar approach was used to demonstrate the existence of phosphorotrithious acid (HS)3P in the dilute gas phase (D189). Holmes and his collaborators have recently published NRMS investigations of C2H7O isomeric radicals and suggested that the stable forms observed involve Rydberg states (D190). The unusual carbon oxides OC2O, OC3O, and C2O, were also reported by Chen and Holmes (D191). The review by Zagorevskii and Holmes (D173) covers work on NRMS of organometallic and inorganic species. An exhaustive study by Holmes and Mayer (D192) of the structures and energetics of cations and radicals of formula (C2, H2, N) incorporated NRMS experiments. An equally ambitious study of the unimolecular chemistry of protonated glycine and its neutralized form, by Wesdemiotis et al. (D193), indicated via NRMS that the charged species must have been a mixture of tautomers (both N and O protonation) at the instant of neutralization. Other investigations of structures and mechanisms for rearrangement and dissociation of small ions and neutrals are quite numerous and require a more detailed review than is possible here. Only a few examples will be mentioned briefly. Distonic radical cations are now firmly established as important structural types in mass spectrometry, and several reviews are available, e.g., (D194-D198). Kentta¨maa has been particularly active in this area, as illustrated by publications (D199-D202) since her 1994 review (D198). Another recent example of a complex mechanism with participation of distonic isomers, in an apparently simple methyl loss reaction, is that described by Burgers et al. (D203). Another class of structures of current interest is that of hydrogenbridged radical cations (D204-D206). Electrochemical Aspects of Electrospray Ionization. The explosive developments in the use of electrospray ionization in mass spectrometry might be dated from a paper by Fenn and his collaborators in 1989 (D207). It is a truism (and therefore true!) to say that the mechanisms at play in electrospray mass spectrometry are complex and still not fully understood. There are four major questions that can be separated out conveniently for purposes of discussion: (1) production of charged droplets at the tip of the electrospray needle; (2) evaporation of solvent from the charged droplets and (possibly) droplet disintegrations yielding extremely small charged droplets capable of producing gas phase ions; (3) mechanism(s) by which ions are ejected from the small liquid droplets into the gas phase; (4) transfer of gaseous ions from the atmospheric pressure conditions of the source to the mass spectrometer vacuum, in such a way that the residual solvent molecules are declustered without dissociating or otherwise altering the analyte ions themselves. The present section is mostly concerned with some new understanding of the first of these questions, which also has implications for the practical applicability of electrospray ionization. It is a common experience that the analytes best suited to electrospray ionization are polar compounds which are readily ionizable in solution prior to entering the electrospray needle. The production of charged droplets from such an (overall electrically neutral) solution is then thought (D208) to be due to electrophoretic separation of positive from negative ions under the influence of the strong electrical field (up to 106-107 V/m).
Nonpolar compounds such as hydrocarbons in nonionic solutions cannot undergo this process and are not usually considered to be amenable to electrospray ionization. However, some nonpolar compounds are susceptible to electrochemical oxidation to their molecular radical cations. Following insights provided by Kebarle concerning the view of an electrospray source as an electrochemical cell of a special kind (D208), Van Berkel (D209-D215) has developed procedures whereby compounds like polycyclic aromatic compounds (PAHs) can be electrochemically oxidized at the electrospray needle, thus providing droplets charged because they contain the resulting PAH cations. Van Berkel has been able to obtain electrospray mass spectra of PAHs and porphyrins, showing excellent signals for the respective molecular radical cations and even in some circumstances for the molecular dications (D215). This work could be regarded as an innovative application, but is included in the Fundamentals section because of the exemplary way in which the technique was developed by thinking through the electrochemical principles in the electrospray context. Space does not permit a detailed account of this development (D209-D215), unfortunately, but some of the more pertinent points can be summarized. A useful model of an electrospray ion source is that of a controlled current electrolytic cell. If we wish to efficiently oxidize (ionize) a PAH, for example, by electrochemical means, the magnitude of the total electrospray current (electrolytic current) must be sufficient for the oxidation of all species in solution that are as easily or more easily oxidized than the PAH. Of course the PAH must be available for oxidation at the solution-needle interface. To satisfy these requirements, Van Berkel et al. showed that addition of a suitable electrolyte to the electrosprayed solution, in order to raise the total current sufficiently, could be combined with lower flow rates (typically 5 µL/min) and use of a platinum rather than a steel needle, to satisfy the theoretical requirements in a practical electrospray source. The key requirement for a suitable electrolyte soluble in the nonpolar solvents used was satisfied by the finding that lithium trifluoromethanesulfonate (“lithium triflate”) could be dissolved to a sufficient extent in mixtures of acetonitrile and dichloromethane. A recognizable mass spectrum (m/z 100-370) was obtained (D214) by electrospray ionization of as little as 270 fmol of perylene. This strategy, developed from considerations of the fundamental physical chemistry appropriate to the problem (D209-D215), is inherently more convenient and simple than the alternative strategy of combining a discrete electrochemical cell upstream from an electrospray source, for the same purpose (D216). PROTEIN MOLECULAR WEIGHT DETERMINATION During this review period there has been a considerable increase in the use of MALDI and ES mass spectrometry just for determination of the molecular weights and purity of natural and recombinant proteins. These reports are summarized in Table 1. PEPTIDES AND PROTEINS As indicated in other sections of this review, there is growing awareness in the biomedical community that mass spectrometricbased techniques have become the methods of choice for handling most types of problems encountered in the characterization of the primary covalent structure of proteins derived from both natural and recombinant sources (A3, A6, F1-F15). The Analytical Chemistry, Vol. 68, No. 12, June 15, 1996
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Table 1. Reports of Peptide/Protein Molecular Weight peptide/protein
species/source
method used
ref
AKH/RPCH family peptide Alzheimer amyloid Aβ peptide variants Alzheimer amyloid Aβ 17-42 peptide amelogenin anti-p185HER2 antibody huMAbD5 apolipophorin III arsenate reductase ATP synthase subunit 6 (ATP6) CA (capsid) protein calcium-binding protein S100A3 calcium-binding proteins (calbindins-D28k) calgranulin C catalase CbiB2 chaperonin 10 chaperonin 10 chaperonins 60 and 10 chromogranin B collagen types I and II creatine kinase cysteine-rich intestial protein (CRIP) cytochrome c553 cytochrome f cytosolic phospholipase A2 (cPLA2) [D-Leu2]deltorphin deoxyhypusine synthase diacetin B divergicin A DNA-binding cI434 repressor DTPA-rhG-CSF conjugate dual-specific protein tyrosine phosphatase dynorphin-processing endopeptidase endothelins EF-hand Ca2+-binding protein enkephalins Fab fragment fatty acid-binding protein Fel dI ferredoxin I ferredoxin VI (FdVI) fibroblast growth factor (bFGF or FGF-2) flavin reductase glucagon-like peptide-1 (GLP-1) glutathione S-transferase (GST 1) glyoxalase I GS-RNase T1 and GS-R-lactalbumin guanylin halocyanin Hb G-Waimanalo hemoglobin A heparin-binding protein histidine tagged Onc M human cytomegalovirus protease IF1 IF3 integrin RIIbβ3 K-carrageenase R-lactalbumin larval and pupal cuticular proteins lanthionine-containing antibacterial peptide SA-FF22 leech-derived tryptase inhibitor LYCP I, II, and III major surface layer protein molluscan egg-laying peptide prohormone mutant interleukin-6 myelin basic proteins non-protein nitrogen old yellow enzyme PA28 peptide V peptidyl-prolyl cis/trans isomerase pheromone biosyntheis activating neuropeptide (PBAN) plastocyanin (mutant) polyketide synthase acyl carrier proteins pancreatic colipase preproenkephalin A-derived peptides proglucagon
Anax imperator mauricianus corpora cardiaca human human mouse human Locusta migratoria Staphylococcus aureus petunia avian sarcoma and leukemia viruses human rat brain pig granulocytes Proteus mirabilis (PR) Carnobacterium piscicola human mouse Thermoanaerobacter brockii chromaffin granules equine human muscle and brain rat small intestine DesulfoVibrio Vulgaris Hildenborough Brassica rapa human Phyllomedusa burmeisteri rat testis Lactococcus lactis subsp. lactis Carnobacterium diVergens synthetic human human bovine pituitary human Orconectes limosus abdominal muscle Theromyzon tessulatum mouse Chaenocephalus aceratus cat dander DesulfoVibrio africanus Rhodobacter capsulatus human, bovine bovine liver human colon Mytilus edulis gill Pseudomonas putida human rat intestinal mucosa Natronobacterium pharaonis human transgenic swine porcine platelets human human cytomegalovirus potato E. coli human platelet Alteromonas carrageenoVora bovine Tenebrio molitor Streptococcus pyogenes strain FF22 Hirudo medicinalis Lymnaea stagnalis Clostridium thermosaccharolyticum Lymnaea stagnalis human human brain Parmigiano-Reggiano cheese Saccharomyces cereVisiae bovine heart bovine posterior pituitary E. coli HelicoVerpa zea spinach Streptomyces porcine bovine pituitary porcine and human pancreas
ES ES PDMS MALDI ES ES ES MALDI ES ES MALDI ES Edman, ES ES ES ES ES LSIMS MALDI ES MALDI ES ES ES LSIMS MALDI ES ES ES MALDI MALDI MALDI LSIMS ES ES ES ES MALDI ES ES ES ES MALDI ES MALDI ES ES ES LSIMS MALDI ES ES ES ES MALDI ES ES ES ES ES ES MALDI MALDI MALDI ES ES FAB ES ES ES ES ES MALDI ES ES ES MALDI
E1 E2 E3 E4 E5 E6 E7 E8 E9 E10 E11 E12 E13 E14 E15 E16 E17 E18 E19 E20 E21 E22 E23 E24 E25 E26 E27 E28 E29 E30 E31 E32 E33 E34 E35 E36 E37 E38 E39 E40 B41 E42 E43 E44 E45 E46 E47 E48 E49 E50 E51 E52 E53 E54 E55 E56 E57 E58 E59 E60 E61 E62 E63 E64 E65 E66 E67 E68 E69 E70 E71 E72 E73 E74 E75 E76 E77
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Table 1 (Continued) peptide/protein
species/source
method used
ref
prodynorphin-derived peptides prophenoloxidase prostate specific antigen (PSA) protamine-like protein (PL-III) protein-hapten conjugates proteinase inhibitor precursors pseudomycins putative zinc finger protein recombinant human granulocity-macrophage colony-stimulating factor RES-701-1, endothelin type B receptor antagonist review review rhamnogalacturonan acetylesterase (RGAE) rhodanese rubredoxin serine hydroxymethyltransferase (SHMT) siderophores signal transduction protein P11 soluble CD14 Stp1 subtilisin protease, recombinant variant tetrameric hemoglobin TGF-β1 transferrin TRAP Tyr-K-MIF-1 tyrocidine synthetase 1 wing-specific cuticular proteins
rat brain Bombyx mori larval hemolymph human Mytilus trossulus sperm bovine Locusta migratoria Pseudomonas syringae cuttlefish epididymal sperm E. coli Streptomyces sp. RE-701 N/A N/A Aspergillus aculeatus C254 mutant Heliobacillus mobilis E. coli Vibrio hollisae and Vibrio mimicus E. coli Chinese hamster ovary Schizosaccharomyces pombe Bacillus human human peripheral neuroepithelioma human Bacillus subtilis human brain cortex Bacillus breVis Locusta migratoria
ES MALDI ES MALDI MALDI ES LSIMS ES ES LSIMS ES, MALDI ES MALDI ES MALDI ES LSIMS MALDI MALDI ES LSIMS ES MALDI ES, MALDI MALDI ES MALDI ES
E78 E79 E80 E81 E82 E83 E84 E85 E86 E87 E88 E89 E90 E91 E92 E93 E94 E95 E96 E97 E98 E99 E100 E101 E102 E103 E104 E105
popularity of these methods is due to the inherent rigor of the mass spectral data which may be obtained at high absolute sensitivity with relative ease. Mass spectral techniques are also rather robust, routine, and rapid for quantities of peptide or protein available in the low-picomole range. In addition, these methods have the advantage of being very versatile, in that most classes of structures may be detected and studied including those which are most unexpected (F16, F17). Since many peptides display incomplete fragmentation upon low energy collisional activation in tandem experiments (F18), high-energy collision-induced dissociation is of clear advantage for de novo peptide sequence determination and oligonucleotide probe design (F19). Attempts to work in the femtomole range pose a variety of problems due to various types of contaminants usually present in biological preparations that may mask the sample (detergent mixtures such as Triton X-100, nonvolatile salts, contaminants of unknown origin, etc.). Eradicating such contaminating substances can be most difficult when tackling new projects not previously subjected to mass spectrometric scrutiny. While MALDI is somewhat more tolerant (F8) than electrospray (F20) to many of the contaminants present in biological isolations, the quality of spectra obtained is usually compromised unless HPLC purification is carried out. This tolerance seems connected to “separations” occurring during the crystallization of the sample with the MALDI matrix during target preparation, and the fact that certain spots (“purified” during sample crystallization) give better signals than others. MALDI is unpredictably selective among the components of an unseparated mixture detected in a given experiment (F21). This situation can be altered by washing sometimes, or by repetitive analyses in different matrices (F21), but again separation of components would be the best approach if feasible. In addition, there may be difficulty in “finding” particular peptides in a protein digest, even from reversed-phase HPLC/electrospray analyses, which may be associated with high solubility of very hydrophilic components or insolubility of hydrophobic components or loss on the column. Of course, in many cases these are
not major issues, unless the missing or poorly behaved peptide(s) happen to be the one(s) that are the focus of the study (viz. whether, where, and/or how a peptide is modified; determination of its phosphorylation sites; sites of cross-linkage; residues bearing O-linked glycosylation; etc.). Finally, in reaching the absolute sensitivity of the instrumentation at the low-femtomole level and below, chemical noise of unknown origin from sample isolation and handling (laboratory dust, solvent contaminants, vial contaminants, etc.) can be a major issue not easily overcome (A18). Of course, without having the high inherent instrumental sensitivity, it would not be possible to “see” such chemical noise to begin with. Thus it would not even be possible to eventually locate the sources of the contaminants and take steps to eliminate or minimize them. Thought will need to be given to use of clean room techniques for very low level sample handling in the near future. Unfortunately, many laboratories do not have all of the most advanced versions of instruments necessary to succeed in tackling any type of problem in protein biology. Such a lack may be more critical in academic research settings that are focused on identification and structural investigation of natural proteins available in limited supply. However, this situation has led to reports in the literature biased by limited experience that can lack balance and objectivity in assessing the actual relative merits of different techniques, approaches, and instrumentation. Varying opinions certainly exist on MALDI versus electrospray ionization, or the quality and information content of tandem mass spectra desirable, for example, where judgments appear based on considerations other than the technical fact. Newcomers to the field should evaluate their needs carefully and invest in instrumentation wisely, since their state-of-the-art lifetimes are presently only a few years at best. Vigorous further development of commercial instrumentation is underway that will provide significantly improved performance using both MALDI and ES ionization (see section C). Synthetic Peptides. Over the last five years the percentage of synthetic peptides prepared by FMOC chemistry has increased. Analytical Chemistry, Vol. 68, No. 12, June 15, 1996
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Recent evaluation of the products of 82 crude preparations from a variety of laboratories participating in the study contained 59% of the desired product as estimated by electrospray and MALDI mass spectrometry and 39% of the desired product as estimated by reversed-phase HPLC (F22). These authors suggest that “proper peptide characterization is essential, especially in light of the lack of correlation between product quality and cleavage reagents or workup protocols”. Mass spectrometry has been employed to establish synthetic peptide fragments in the case of neuropeptide Y analogues (F23), photoreactive benzophenonecontaining analogues of parathyroid hormone (F24), R-factor mating pheromone of S. cerevisiae (F25), and HSV-1 gD 268-284 and IL-6 179-185 (F26). Preparation of an Edman-type protein-sequencing reagent 4-(3pyridinylmethylaminocarboxypropyl)phenyl isothiocyanate has been shown to bring detection levels to the low-femtomole range using electrospray mass spectrometry (F27, F28). Use of anhydrous hydrazine vapor for chemical cleavage of asparaginyl and glycyl-glycine peptide bonds has been reported (F29). Tryptophan-specific protein cleavage has been evaluated using BNPS-skatole (F30). Incorporation of 18O into a peptide during isoaspartyl repair demonstrates repeated passage through a succinimide intermediate in insuring that a stoichiometric product is obtained (F31). N-terminal degradation of recombinant human growth hormone can occur with the formation of a diketopiperazine (F32). Similar loss of the first two residues of the amino terminus of human serum albumin occurs when stored at or above 30 °C (F33). The procedure for conversion of the N-terminal of a peptide or protein into a 2-oxoacyl group by nonenzymic transamination and its subsequent removal with phenylene-1,2diamine has been reported (F34). Trifluoroethyl isothiocyanate has been prepared for use as a volatile Edman reagent for peptide ladder sequencing by MALDI mass spectrometry (F35). Toxins, Neuropeptides, and Antigens. The primary structures of two proteins from the venom of the Mexican red knee tarantula Brachypelma smithii have been reported (F36). Edman degradation and tandem mass spectrometry were used in the analysis. Protein 1 was shown to contain 39 residues, including the identification of six cysteines as three disulfides. Protein 5 was shown to contain 34 residues, including six cysteines as disulfides. In another report, a 34-residue cationic and amphiphatic peptide designated dermaseptin I was isolated from the skin of arboreal frog Phyllomedusa sauvagii that exhibits microbicidal activity against bacteria, yeast, protozoa, and filametous fungi (F37). This work was accomplished by solid-phase Edman degradation and measurement of molecular weights by electrospray mass spectrometry. A particularly interesting peptide isomerase has been reported from the venom of funnel web spider Agelenopsis aperta (F38). This isomerase is a disulfide-linked heterodimer which interconverts the L to D configuration of serine-46 of the 48-amino acid peptide ω-agatoxin-TK. A variety of mass spectrometric methods together with Edman degradation have been employed to establish the structures of conotoxins from Conus pennaceus (F39), a mollusc-specific R-conotoxin, and µPnIVA and µPnIVB (F40), two novel sodium channel-blocking peptides which possess a new cysteine residue framework for conotoxins. Revision of the structure of a cysteinerich conotoxin, TxVIIA, containing γ-carboxyglutamic acid residues has been reported from the sea snail Conus textile (F41). 626R
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This conotoxin bears the conserved cysteine framework for ωand δ-conotoxins. Several mass spectrometric methods were employed to establish the presence and location of the γ-carboxyglutamic acid residues in this conotoxin, including electrospray, MALDI in both positive and negative ion modes, and in particular high-energy CID analysis using MALDI ionization on an EBEorthogonal time-of-flight tandem instrument (C80). There is evidence from this work that Gla-containing peptides may be detected by simply comparing the molecular weights observed in positive ion and negative ion mode MALDI with electrospray mass spectra, since essentially quantitative decarboxylation of the γ-carboxy functions takes place in the negative ion mode in MALDI. A preliminary report has appeared on the analysis of conotoxin venoms from several species using LC/electrospray mass spectrometry (F42). Electrospray mass spectrometry was used to characterize the products of limited proteolysis of Sh I, a polypeptide neurotoxin from a sea anenome (F43). Neuropeptide expression and processing in single neurons from Lymnaea brain has been carried out by MALDI (F44, F45). Mass spectrometric methods continue to be employed in the characterization of MHC peptides from both class I and class II molecules. These projects include the definition of specific peptide motifs for four major HLA-A alleles (F46), HLA-A2 subtypes (F47), HLA-B*4402 and -B*4403 (F48), and HLA-DQ1 and -DQ8 associated with type 1 diabetes (F49). MALDI has been employed to characterize the molecular weight distribution of a subset of peptides from HLA-B27, revealing a population of peptides ranging from 900 to 4000 Da in size (F50). Protein Mass Mapping. Shackleton and Witkowska have provided a recent overview of the use of mass spectrometric methods for the characterization of human hemoglobin variants, estimated to be carried by 150 million people worldwide (F51). They note that one of the most difficult challenges in future routine clinical diagnoses will be in establishing unambiguously the absence of hemoglobinopathy in a rapid, cost-effective manner. The complete sequence of hemoglobin from the marine polychaete tail of Tylorrhynchus heterochaetus has been published (F52). In addition to validating the sequences of three of the four globin chains and two linker chains, the presence of three additional linkers was established. The resulting calculated mass of this complex was found to be 3.42 MDa, close to the observed sedimentation equilibrium value of 3.38. The software MaxEnt was of considerable value in the processing of the electrospray mass spectrometry data in order to obtain sufficient mass resolution of all the chains and subunits in this large heteromultimeric protein complex. The properties of a recombinant human hemoglobin D99(β)K have been reported (F53). Studies of histone H5 from Xenopus laevis erythrocytes reveal three posttranslational modifications, including N-terminal acetylation, acetylation of Lys-16 and -12, and dimethylation of Lys-20 (F54). Another posttranslational modification, phosphorylation, has been studied in ram spermotidyl protamine (F54). Determination of the sequence of the largest known protamine isolated from the sperm of the archaeogastropod Monodonta turbinata made effective use of electrospray methods (F55). Studies of familial Alzheimer’s disease brain amyloid β protein species reveal that the APP717 missense mutation affects the ratio of Aβ1-42/ 43 and Aβ1-40 (F56).
Electrospray mass spectrometry has been employed in studying the conversion of proparathyroid hormone to parathyroid hormone by the prohormone convertase furin (F57). Studies of plasma tissue factor pathway inhibitor by electrospray mass spectrometry revealed that a substantial portion of the carboxy terminus, including the third Kunitz-type domain, is missing in its association with low-density lipoproteins (F58). Structural analysis using electrospray mass spectrometry confirmed that norleucine residues replaced all four methionine positions in recombinant construct of human macrophage colony stimulating factor (F59). Electrospray mass spectrometry has also been used to characterize the products of limited proteolysis of human apolipoprotein A1 (F60). A 10 kDa protein analogous to GroES from armadillo tissues infected with Mycobacterium leprae has been characterized and expressed (F61). Peptides obtained by cyanogen bromide cleavage of the rat seminal vesicle epithelial protein SV-IV at Met-70 were assayed as possible transglutaminase substrates in addition to being evaluated for possible antiinflammatory, antithrombotic, and immunosuppressive properties (F62). The colicin E9 sequence has been mapped by electrospray mass spectrometry (F63). Electrospray mass spectrometry has been used to characterize the sites of proteolysis of ATP synthase derived from bovine heart mitochondria (F64). Mass spectrometric characterization of the complex of active site inhibited factor VIIa and soluble tissue factor (sTF) indicated the presence of sTF fragments similar to those formed by proteolytic digestion with subtilisin (F65). Electrospray mass spectrometry was employed to characterize the protease processing sites in a multidomain protease inhibitor precursor from Nicotiana alata (F66). The flavoprotein EpiD has been shown to carry out oxidative decarboxylation of the precursor peptide (F67). Plasma desorption mass spectrometry was employed to identify sites of modification of recombinant human PAI-2 with putrescine catalyzed by guinea pig liver transglutaminase (F68). PDMS was also employed to characterize the proteolytic fragments of the isoforms of Bet v 1, a major birch pollen allergen (F69). From studies of tetranitromethane protein modification, evidence has been presented that tyrosine-57 in the NH2-terminal domain of uPAR and tyrosine-24 in uPA are engaged in the receptor-ligand interaction (F70). Several posttranslational modifications of watersoluble human lens crystallins from young adult donors have been identified using electrospray mass spectrometry (F71). In addition, deamidations were revealed in the water-soluble portion of crystallins from lenses of older donors (F72). MALDI has been employed to verify the sequence of E. coli isoleucyl tRNA synthetase (F73). Electrospray mass spectrometry has been important in the characterization of 10 subunits of bovine cytochrome bc1 (F74). Electrospray was also critical in identifying species that appear to be cytochrome b562 holoproteins with thioether covalent linkages to heme (F75). -N-Acetyllysine has been found in recombinant porcine and bovine somatotropins synthesized in E. coli (F76). The presence of N--methyllysine residues has been identified in several proteins, including aspartate aminotransferase from Sulfolobus solfataricus (F77), amphioxus troponin C (F78), and ferredoxin-NADP reductase from Chlamydomonas reinhardtii (F79). The site of covalent attachment of FAD to the berberine bridge enzyme has been shown by MALDI to be His-104 (F80). Electrospray mass spectrometry has been used to establish the modification of cytochrome c with DOTA (1,4,7,10-tetraazacyclododecane N,N′,N′′,N′′′-tetraacetic acid) (F81).
This class of chelating agents forms stable complexes with a variety of radionuclides important in radioimmunoimaging and immunotherapy. The human homologue of Schizosaccharomyces pombe Rad2 protein has been established as a nuclear 5′,3′-exonuclease using MALDI analysis of proteolytic digests of the enzyme purified from HeLa cells (F82). Extensive studies of the proteolysis of recombinant human estrogen receptor purified as a complex with estrodiol E. coli required characterization by HPLC/electrospray mass spectrometry (F83). From these studies, it appears that the structural N-terminus of the hormone binding domain begins with a tightly wound region at N304. From this data the C-terminal region was less clearly defined. Expression of murine interleukin-6 in E. coli revealed a population of C-terminally truncated analogues extended with a 10Sa RNA decapeptide (F84). This recombinant protein extension chimera appears dependant on the 10Sa RNA gene. HPLC/electrospray mass spectrometry was employed to verify the sequence of DNA polymerase expressed in Thermus aquaticus (F85). Verification of the sequence and disulfide bonding of Pseudomonas exotoxin 40 secreted from E. coli has been accomplished using electrospray mass spectrometry (F86). Covalently Modified Protein N- and C-Termini. Mass spectrometry clearly has become the technology of choice for dealing with modified protein sequence. From the outset it has been recognized that mass spectrometry is the most effective technique for elucidation of the nature of blocked amino termini. As in earlier reviews, there are a variety of recent examples of the use of tandem mass spectrometry and related methods for this purpose. High-energy collision-induced dissociation provides clear information on the sequence and nature of N-terminally blocked peptides. Current examples include determination of N-terminal acetylation in microsomal glutathione S-transferase (F87), both cytoskeletal and fibroblast nonmuscle type tropomyosins (F88, F89), and stathmin (F89) from melanoma A375 cells. In addition, glutathione S-transferase from Schistosoma mansoni has been shown to be N-terminally acetylated (F90). Several other reports of N-terminal acetylation include the β-galactoside binding lectin, galectin-5 (F91), the S-100 related calcium-binding protein isolated from bovine retina, Cap1 (F92), a phosphotyrosine protein phosphatase from porcine liver (F93), recombinant annexin V expressed in S. cerevisiae (F94), the squid retinalbinding protein (F95), and the glucose 6-phosphate dehydrogenase from human erythrocytes (F96). Two proteins have been shown to be blocked by pyroglutamic acid-endocuticular protein from two locust species (F97), and human filaggrin (F98). Acylation of protein N-termini is a well established posttranslational modification, but recently heterogeneous N-acylation has been characterized on the cAMP-dependent protein kinase derived from bovine retina in a manner similar to earlier reports on recoverin and transducin (F16). This heterogeneity appears tissue specific, since cAMP-dependent protein kinase isolated from heart and brain bore only myristate (F16). N-terminal heterogeneity was revealed by MALDI in recombinant fatty acid binding protein from bovine heart expressed in E. coli (F99). A drawback of peptide sequencing using the Edman degradation method is that there is virtually no way of knowing when or whether the C-terminus has been reached in fact. Mass spectrometry provides unambiguous information in this regard. An interesting example concerns the nature of arrestin isolated from Analytical Chemistry, Vol. 68, No. 12, June 15, 1996
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rod outer segments (F100). The amino acid sequence available was shown to be identical with arrestin, except that the 35 C-terminal residues, positions 370-404, were replaced by a single alanine residue, apparently generated by alternative mRNA splicing (F100). Electrospray mass spectrometry provided information on the C-terminal truncation of three isolectins of soybean agglutinin (F101). Characterization of class I and IVa β-tubulin isotypes by mass spectrometry established that brain-specific class IVa and constitutive class I isotype can be glutamylated at Glu-434 and Glu-441, respectively (F102). Studies on tubulin from porcine brain have also been reported (F103). In contrast, tubulin isolated from Paramecium axonemal tubulin has been shown to be modified by polyglycylation containing up to 34 glycyl units covalently bound to the γ-carboxyl group of glutamyl residues (F104). The complete amino acid sequence of the Aa6 subunit of the scorpion hemocyanin from Androctonus australis has been reported (F105). Polyphenolic proteins in the adhesive plaques of the the marine mussel, Mytilus edulis, have been shown to contain a new residue, hydroxyarginine, by high-energy CID analysis (F106). Studies on the self-inactivation of mammalian 15-lipoxygenases have established that Met-590 in the human enzyme and Met-591 in the rabbit enzyme are oxidized, but appear nonessential for inactivation (F107). It is suggested that this methionine might reside in the substrate binding pocket and interact with the enzyme product. Electrospray mass spectrometry has provided a delineation of microheterogeneity in the lectins Jacalin and Maclura pomifera agglutinin (F108). High-energy CID analysis has been used to establish the primary structure of thioltransferase from human red blood cells (F109). This revealed that the N-terminal peptide was acetylated. Low-energy CID was used in the sequence analysis of mouse liver NAD(P)H:quinone acceptor oxidoreductase (F110). Work continues on the development of more sensitive methods for peptide sequence analysis. Recently, considerable attention has been turned to the type and quality of information which can be obtained from metastable fragment analysis, so-called postsource decay, upon MALDI using a reflectron time-of-flight instrument. A recent detailed comparison of the qualitative nature of peptide fragmentation obtained by MALDI reflector time-offlight with that obtained from LSIMS tandem four-sector highenergy collisional activation has been reported (F111). In addition, it has been shown that high-energy collisional activation of MALDI-generated ions produces spectra which are qualitatively comparable to four sector CID data (F112). Thiols and Disulfide Bonds. Mass spectrometry has been employed for more than a decade in establishing disulfide bonding assignments in proteins. A variety of reports have appeared, ranging from confirmation of disulfide bond formation during synthesis (F113), through selective thiol modifications and assignment of complex disulfide linkages in various proteins. In studies of superoxide dismutase by HPLC/electrospray mass spectrometry, performic acid oxidation was employed to assist in protein unfolding, to allow more complete digestion and create additional cleavage sites for endoproteinase Asp-N. In addition to the anticipated oxidation of cysteine cross-links to cysteic acid residues, peptides were formed with molecular masses 66 and 100 Da higher than calculated for a missing expected peptide. Using model peptides of similar sequence, further tandem mass 628R
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spectrometry analyses were carried out that established that the N-terminal methionine in the model peptide was oxidized to methionine sulfone with an increase of 32 Da, whereas the tyrosine residue showed an increase of either 34 or 68 mass units. From accurate mass measurement, the atomic composition was deduced. These measurements revealed that tyrosine was electrophilically substituted by one and two chloro substituents, respectively, presumably arising from the sodium chloride used in the buffer of the oxidizing medium (F114). Mass spectrometry has been employed to establish that amino acid 47 in SP-22, a substrate for a mitochondrial ATP-dependent protease in bovine adrenal cortex, is in fact cysteine-sulfinic acid (F115). In studies of catalase HPII of E. coli, cysteine-438 was found to be modified by 43 ( 2 Da, thought to represent a hemithioacetal (F116). Electrospray was used to establish the modification of a surface reactive cysteine residue of the class-II fructose-1,6-bisphosphate aldolase of E. coli as a covalent adduct with 2-mercaptoethanol, thereby adding 76 Da (F117). 5-Iodoacetamide fluorescein (5-IAF) was used to probe the reactive cysteines involved in S-adenosyl-L-methionine binding in human soluble catechol O-methyltransferase expressed in E. coli (F118). MALDI was employed to demonstrate that histidine-229 at the active site of sorghum leaf NADP-dependent malate dehydrogenase was modified by diethylpyrocarbonate (F119). Mass spectrometry was used to analyze the refolding intermediates of recombinant human macrophage-colony-stimulating factor (F120). Eleven species of scrambled disulfide structures in recombinant hirudin have been identified by mass spectrometry (F121). Recombinant intestinal trefoil factor from rat and human have been produced in S. cerevisiae. Of seven cysteine residues in the monomeric protein, six exist as disulfide bridges, while the seventh, cysteine-57, forms a cystine cross-link dimer (F122). Studies of a protein modeled after a core repeat in an aquatic insect’s silk protein revealed the presence of confirmational isomers differing in their intramolecular disulfide bonding topologies (F123). Mass spectrometry was involved in establishing that a disulfide bond exists between cysteine-379 residues of adjacent von Willebrand factor subunits (F124). The amino acid sequence and disulfide bond assignments have been reported for oat alcoholsoluble avenin-3 (F125). Mass spectrometry was again employed to establish the intramolecular disulfide-bonded loops in the extracellular domain of human natriuretic peptide receptor-C (F126). Both electrospray and MALDI were used to identify the sites of intra- and intermolecular disulfide linkages in bovine dopamine β-hydroxylase (F127). An extra sulfur atom has been reported in the Cys-182-Cys-189 cystine bridge in recombinant human growth hormone produced in E. coli (F128). A system has been devised that permits the efficient substitution of cysteine residues in a protein by selenocysteine. In structural studies by electrospray mass spectrometry, the occurrence of diselenide, selenosulfur, and disulfide bridges were established for the first time (F129). During studies of the solution structure of the antifungal protein AFP from Aspergillus giganteus by NMR, evidence for cysteine pairing isomerism involving the four disulfide bridges has been supported by mass spectrometric measurements (F130). An interesting report has provided evidence that single intermolecular disulfide-bonded peptides can be fragmented at the S-S bond by increasing the laser fluence during MALDI mass spectrometric analysis (F131).
Gels, Mass Mapping, Sequencing, and Database Interrogation. The use of gels for the separation of macromolecules was first reported in 1954 (F132) and expanded in power through the addition of an isoelectric focusing dimension by O’Farrell in 1975. Since that time, much of the effort using such gel analyses has focused on differential mapping of gene expression at the protein level through analysis of the coordinates and relative abundances of stained spots on 2D SDS PAGE systems (F133, F134). Considerable effort has been expended on development of 2D gel protein spot coordinate reproducibility and development of computer programs for the archiving and comparison of 2D gel patterns. However, protein spot identification using Edman microsequencing has been challenging even in the best of circumstances. This has been due to a combination of lack of sufficient protein quantities in Coomassie-stained 2D gel spots, the widespread occurrence of N-terminally blocked proteins, poor protein recoveries from gels, and other unknown factors. The situation has changed dramatically in the last two to three years through the application of the power of mass spectrometric methods to effect unambiguous protein identification of Coomassie-stained protein spots (A3, F135, F136). In addition, progress in the mapping, tagging, and sequencing of the human genome has proceeded at an unexpectedly rapid pace (F137), such that the scientific community is on the verge of entering the post-genome era as this is being written. Thus, with the imminent prospect of having the human and other genomes available as giant computer look-up tables, it becomes feasible to exploit the inherent speed and precision of mass spectrometric methods for identification of cell proteins on the scale required to define gene expression in any given cell type for the first time. It would be prudent as the development of mass spectrometric strategies evolves to preserve an inherent ability to obtain de novo amino acid sequence, rather than settle for simple mass mapping of molecular weights of proteolytic digests of 2D gel proteins, despite the fact that mass mapping and database searching is adequate in many cases for spot identification. Tandem mass spectrometric sequence information will certainly speed the next phase involving the elucidation of the physiologically active forms of proteins and their interconversions during activation and deactivation (F138-F147). Patterson and Aebersold have recently provided a comprehensive review of mass spectrometric strategies for the identification of proteins separated by gel electrophoresis (F148) and have prepared a forthcoming chapter on current problems in protein biochemistry and their technical solutions (F149). Tempst and co-workers describe protocols for the sequence analysis of less than 5 pmol of peptides using reversed-phase HPLC, chemical sequencing, and MALDI mass spectrometry (F150, F151). This strategy was applied to identify the gel purified 250 kDa protein that is the mammalian target of rapamycin-FKBP12 complex (F152). MALDI has been employed to determine the sites of phosphorylation of snapsin Ia and Ib (F153). The identification of tyrosine phosphorylated proteins from B lymphocytes stimulated through the antigen receptor has been described (F154). Numatric/B23/nucleophosmin has been identified in apoptotic Jurkat T-lymphoblasts (F155). MALDI was employed to establish that the 43 kDa protein found in Shc immunoprecipitates was actin (F156). Different isoforms of the hypusine-containing protein, eIF-5A, from HeLa cells revealed that lysine-47 was acetylated, and lysine-50 modified by hypusine (F157). In the course of study
of isoforms of a cuticular protein from larvae of a meal beetle, Tenebrio molitor, mass spectrometry revealed that the N-terminus was modified by acrylamide (F158). Electrospray mass spectrometry was used to show that multiple banding of β-lactamase on isoelectric focusing gels was due to the loss of a different number of N-terminal amino acids (F159). As discussed elsewhere in this review, considerable improvement in low-level sample utilization efficiency has become possible through the development of much lower flow rates for electrospray ionization mass spectrometry (F160). Low-nanoliter per minute rates have been applied to the identification of a heterogeneous nuclear ribonuclear protein, P2 (F161), and an endothelial cell growth inhibitor (F162). The advantages of nanoliter per minute electrospray include the possibility of minimization of sample consumption or, alternatively, gaining sufficient time to record numbers of low-energy CID spectra to improve ion statistics from triple quadrupole mass analyzers. This method was recently applied to obtaining some sequence information from standard proteins isolated from silver-stained polyacrylamide gels (F163). Recent experiments using a capillary HPLC nanoflow per minute electrospray triple quadrupole system established that high-quality mass spectra and in-source fragment ion generation can be obtained using only a 6 fmol aliquot of fetuin. In addition to facile detection and analysis of the peptides, the three N-linked glycopeptides in the fetuin digest also gave excellent spectra (F164). Finally, work on obtaining molecular weights of 2D gel separated proteins has continued (F165, F166). It should be noted that de novo unambiguous peptide sequencing from 2D gel proteins can be carried out by tandem mass spectrometry in the high-energy CID mode using both liquid SIMS (F135), and MALDI ionization (F167). Hence, it is certainly not necessary that protein sequence be already in a sequence database (F168) in order to use mass spectrometry effectively for unambiguous sequencing and cloning of unknown proteins (F19, F112). Phosphorylation. Protein phosphorylation events are inextricably linked to signal transduction processes, providing sites for protein docking, control of enzymatic activity, and so on. Phosphorylation appears to be important even at low stoichiometries among multiple sites of possible phosphorylation by kinases of different specificities. Methods permitting clear analysis of multiply phosphorylated peptides at various stoichiometric levels are of considerable importance in deciphering these types of protein transformations. One of the most important practical developments during this period is the logical followup to the work of Carr and co-workers using cone voltage fragmentation and electrospray for the generation of moiety-specific ions that can be monitored during a HPLC/ electrospray MS run, originally reported for the detection of glycopeptides (F169). In the case of phosphorylated peptide detection, negative ion orifice potential stepping is carried out monitoring m/z 79, a phosphate-derived ion (F170), illustrated with β-casein and a construct from pp60c-src. This technique was described further by Bean et al. (F171) and used by this group for the selective detection of phosphopeptides that were substrates in synthetic peptide libraries for protein kinase activities (F172). Once phosphopeptides have been detected in a digest, tandem mass spectrometry provides an important general methodology for the determination of phosphorylation sites occupied (F173F176). Low-energy CID analysis has been used for the analysis of 21 phosphorylation sites of rat profilaggrin (F173); the v-MosAnalytical Chemistry, Vol. 68, No. 12, June 15, 1996
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catalyzed phosphorylation sites and autophosphorylation sites on MAP kinase kinase (F174); and a variety of synthetic mono- and diphosphorylated peptides which correspond to the peptides from the C-terminal region isolated after enzymatic digestion of rhodopsin (F175). In addition, high-energy CID analysis has been used to define the phosphorylation sites on recombinant protein kinase C (F176). Mass mapping in connection with Edman degradation and phosphatase treatment continues to be used for studies of phosphopeptides. Mass spectrometry has been involved in studying mitogen-activated protein kinase phosphorylation of MAP kinase kinase (F177); phosphorylation of the 61 kDa calmodulinstimulated cyclic nucleotide phosphodiesterase (F178); the control of rhodopsin multiple phosphorylation (F179); and the control by protein kinase C-mediated phosphorylation at specific sites in tubulin microtubule binding region (F180). Tandem mass spectrometry was used to assign threonine-87 and serine-152 of GAP-43 (F181). Several papers have employed electrospray ionization to determine sites of tyrosine phosphorylation (F182-F184). The protein tyrosine kinase ZAP-70 was shown to have a primary autophosphorylation site at tyrosine-292, and a secondary site at tyrosine-126 (F182). Using HPLC/electrospray mass spectrometry, 14 O-glycoforms and three phosphoforms of modification were shown to be responsible for the heterogeneity of bovine κ-casein caseinomacropeptide (F185). The R and δ isoforms of the 14-3-3 protein family are the phosphorylated forms of Rafactivating 14-3-3 β and ζ at serine-185, a consensus sequence motif for proline-directed kinases (F186). The sarcoplasmic reticular calcium release channel is shown to be phosphorylated at serine2843 in intact rabbit skeletal muscle (F187). Studies of the in vivo phosphorylation site of hexokinase 2 from S. cerevisiae have been reported (F188). Mass spectrometry revealed an unusual dephosphorylated form of OmpR, an osmoregulatory DNA-binding protein of E. coli (F189). Additional studies include ovine Rs1caseins (F190); detection of inherent phosphatase activity in the SH2 domain of human pp60c-src (F191); myelin P0 glycoprotein (F192); human τ isoforms (F193); substrates for wheat germ calcium-dependent protein kinase (F194); bovine β-casein (F195); fatty acid-binding protein from rat mammary epithelial cells (F196); rabbit skeletal muscle RR-tropomyosin (F197); ribosomal protein S7 in Tetrahymena (F198); Rs1-casein from bovine milk (F199); and the monitoring of protein kinase and phosphatase reactions with peptide-phosphopeptide mixtures (F200). Glycosylation. Protein glycosylation has been reviewed in some detail recently by Settineri and Burlingame (F201), and Burlingame (F202), and will not be further reviewed at this time. However, the discovery of a new type of posttranslational modification is noteworthy, involving the C-glycosylation of a specific tryptophan residue in human Rnase Us (F203). Another interesting modification involves serum-derived hyaluronan and associated proteins, the heavy chains of inter-R-trypsin inhibitor. The complex was isolated from pathological synovial fluid from human arthritis patients and characterized by electrospray and collision-induced dissociation. It was found that the C-terminal aspartic acid of each heavy chain was esterified to the C6-hydroxyl group of an internal N-acetylglucosamine of the hyaluronan chain (F204). This appears to be the first demonstrated evidence of the covalent binding of proteins to hyaluronan. 630R
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An interesting method has been reported for locating the sites of O-GlcNAc modified peptides by their collision-induced dissociation after removal of the O-GlcNAc by alkaline β-elimination, converting glycosylserine to 2-aminopropenoic acid (F205). Finally, it should be mentioned that use of delayed extraction and MALDI mass spectrometry of glycopeptides and glycoproteins may obviate some of the earlier problems with sialic acid lability during MALDI (F206). Lipidation. Electrospray was used to establish that the N-terminus of hisactophilin was modified by myristic acid (F207). In a similar fashion, it has been shown that the major substrate of protein kinase C can be demyristoylated by the cytoplasmic fraction of brain synaptosomes (F208). Electrospray ionization using suitable organic solvent has been employed for the characterization of hydrophobic biscysteinylpalmitoylated lung surfactant SP-C protein (F209). The palmitoylation of internal lysines has been established in adenylate cyclase toxin from Bordetella pertussis (F210) and the hemolysin of E. coli (F211). Localization of the palmitoylation site in the transmembrane protein p12E of Friend murine leukaemia virus has been reported (F212). Geranylgeranylation of the adjacent cysteines in the small GTPases Rab1A, Rab3A, and Rab5A have been established by electrospray mass spectrometry (F213). Geranylgeranylation of the γ2 subunit of bovine brain G proteins has also been established by MALDI and electrospray techniques (F214, F215). Evidence has been reported that flavin-containing monooxygenase may catalyze the S-oxidative cleavage of farnesylcysteine and farnesylcysteine methyl ester (F216). Mass spectrometry has been used to evaluate the lipoyl domains of recombinant dihydrolipoyl acetyltransferase of the human pyruvate dehydrogenase complex (F217). Diphytanylglycerylated proteins have been identified in Halobacterium cutirubrum (F218). Covalent Modifications. Lysine residues 67, 84, and 140 have been identified as the sites of in vivo glycation of rat liver aldehyde reductase (F219). Electrospray has been used very effectively for studies of acylenzyme intermediates (F220-F222). In addition, studies of the progressive inactivation of porphobilinogen deaminase by the thiophilic reagent N-ethylmaleimide has been reported (F222). High-energy CID analysis has been used to establish the selective alkylation of cysteine residues by the anti-cancer agent melphalan in metallothionein (F223). Mass spectrometry has been employed for the identification of the domains on the 33 kDa protein that is shielded from NHSbiotinylation by photosystem II (F224). Reports of the alkylation of oxytocin (F225) and thioredoxin (F226) by a dihaloethaneglutathione conjugate have been described. It has been shown that electrospray mass spectrometry is able to monitor reactions of nitric oxide with peptides and proteins (F227). Characterization of peptide-acetaldehyde adducts (F228) and protein 4-hydroxy-2-nonenal adducts (F229) have been reported. Tandem mass spectrometry has been used to identify labeling of active site residues in glycosidases (F230-F232). Mass spectrometry has also been used to identify the modification sites of proteins exposed to the arginine-specific reagent phenylglyoxal (F233). Electrospray CID analysis has revealed the serine residue at the active site of the herpes simplex virus type 1 protease (F234), and high-energy CID analysis has revealed the sites of modification of the HIV protease after treatment with irreversible inhibitors (F235, F236). Mechanism-based inactiva-
tion of cytochrome P450 2B1 by 9-ethynylphenanthrene (F237, F238) and 2-ethynylnaphthalene (F239) has been studied by MALDI. A novel thioester-linked 4-hydroxycinnamyl chromophore has been reported in photoactive yellow protein (F240). MALDI has been used to study the covalent modification of recombinant stem cell factor by poly(ethylene glycol) (F241). The peptide Lys-Phe-Lys, residues 590-592, has been identified as the site of photolabeling of chloride ion transport by 5-nitro-2-(3phenylpropylamino)benzoic acid (F242). Photochemical cross-linking between native rabbit skeletal troponin C and benzoylbenzoyl-troponin I inhibitory peptide has been reported (F243). Cross-linking of Prohead II between lysine and asparagine residues has been established during bacteriophage HK97 head assembly (F244). Two chemical cross-links in the catalytic region of human complement protease C1s by water-soluble carbodiimide have been determined by mass spectrometry (F245). Mass spectrometry has been used to analyze the UV-catalyzed cross-linking of E. coli uracil-DNA glycosylase to DNA (F246). Metal Ion Binding. Further reports on the use of both electrospray and MALDI have appeared, aimed at determining the stoichiometry of metal binding to proteins. AMT1, a transcription factor required for copper-induced expression of metallothionein genes in the yeast Candida glabrata was shown to consist of a uniform species of Cu4Zn1AMT1, a conformer that is competent to bind DNA promoter sequences upstream of MT genes (F247). Mammalian prion protein contains three or four copies of an octapeptide repeat sequence, PHGGGWGQ, in the N-terminal domain. Peptides containing either three or four copies of this octa-repeat peptide sequence were synthesized and probed using MALDI for their ability to bind metal ions. Selectivity for binding of copper ions has been shown, suggesting the possible involvement of aberrant copper metabolism as a factor in the pathogenesis of prion diseases (F248). Electrospray mass spectrometry was used for the determination of the calcium-binding stoichiometry for bovine calmodulin, R-lactalbumin, rabbit parvalbumin, and human stromelysin (F249). Negative ion electrospray was used to show the association of eight atoms of both iron and sulfur with apoferredoxin II from Rhodobacter capsulatus (F250). Similar studies have been reported on molecular variants of a [Fe-2S] ferredoxin from Clostridium pasteurianum (F251). Interactions: Proteins and DNA. Further development of mass spectrometric methods for mapping of protein epitopes and protein-protein interactions have been described involving mass mapping of fragments bound to antibodies as linear epitopes, ligand receptor, or protein-oligonucleotide interactions. This was reported for melittin and glucagon-like peptide-1, and the antibodies which were raised against these two peptides (F252) using MALDI. In more recent work, this strategy was applied to mouse monoclonal antibody 11.1 to define the binding epitope of human basic fibroblast growth factor (F253). This strategy also has been employed for studies of transcription factors that are members of the basic/helix-loop-helix/zipper family of DNA-binding proteins (Max) (F254) and the ribosomal protein L7 (F255). Further studies of protein-nucleic acid interactions as noncovalent complexes have been reviewed recently (F256-F258).
OLIGONUCLEOTIDES AND NUCLEIC ACIDS The capability for mass spectrometric analyses of oligonucleotides has generally lagged behind that of oligopeptides. Whereas major advances in peptide and protein analyses were made in the 1980s using fast atom bombardment/liquid secondary ion MS, success in the mass spectrometric analysis of intact oligonucleotides was limited to a few laboratories [notably that of Grotjahn (G1) (and references there cited)]. During the last few years, however, this position has changed markedly with the effective use of both electrospray ionization and matrix-assisted laser desorption/ionization. Recent aspects of these developments are discussed in this section. The emphasis here is on the analysis of unmodified oligonucleotides; the use of mass spectrometry in the characterization of nucleic acids modified by interaction with environmental toxins is reviewed in section H. The current state of the art in mass spectrometric analyses of oligonucleotides and nucleic acids has been described by McCloskey and co-workers in an excellent review published in Current Opinion in Biotechnology (G2). The value of the review is enhanced by the inclusion of an extended bibliography incorporating critical comments, a feature that is characteristic of this journal. A tutorial account of the application of mass spectrometry to the characterization of nucleic acids (at both oligomer and monomer levels) has been presented by Crain (G3). Pomerantz et al. (G4) have assessed the value of molecular mass data alone in establishing oligonucleotide composition. If the compositional value for one residue is known (as, for example, following hydrolysis of RNA by ribonuclease T1, which yields oligonucleotides incoroporating one phosphorylated guanosine residue) then determination of molecular mass to within 0.01% (frequently achievable using electrospray MS on a quadrupole instrument) uniquely specifies base composition to the 14-mer level. Substantial progress has been made in the last two to three years in the application of MALDI (most commonly with a TOF analyzer) to the analysis of oligonucleotides (G5, G6) so that investigation of, for example, a 50-mer is now somewhat routine and there are individual reports (see below) of the analysis of much larger RNAs. Chemical modifications may also be effectively studied (G7). The benefits of delayed extraction (section C) to the analysis of single-stranded DNA (27- to 68-mers) by MALDI/TOF MS have been illustrated by the work of Reilly and co-workers (G8). The success of MALDI analyses is strongly dependent on sample preparation (specifically, the removal of excessive quantities of salts), the choice of matrix, and the oligonucleotide sequence (G9). Detection in the negative ion mode is more commonly used but is not universal. Kirpekar et al. (G10), for example, detected RNA up to 150 kDa (461 nucleotides) in the positive ion mode, and Keough et al. (G11) preferred positive ion analyses of methylphosphonate oligodeoxyribonucleotides designed for evaluation of antisense nucleotide therapies. Nordhoff et al. (G12) evaluated matrices for UV and IR MALDI with detection of negative ions; mixtures of 2-aminobenzoic acid and nicotinic acid were favored for UV MALDI whereas succinic acid (or nicotinic acid for larger RNA molecules) was preferred when an IR laser was used. Fitzgerald et al. (G13) surveyed a large number of basic matrices for UV MALDI, concluding that 2-amino-5-nitropyridine was particularly effective for the analysis of smaller oligonucleotides (less than 20 residues) and for homopolymers of thymidine. The introduction of 3-hyAnalytical Chemistry, Vol. 68, No. 12, June 15, 1996
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droxypicolinic acid as a matrix by Wu et al. (G14, G15) is generally considered to have represented a major advance. At 355-nm desorption wavelength, the yields of positive and negative ions were similar. Finally, Lecchi et al. (G16) suggest further improvements (with respect to reproducibility and the achievement of satisfactory resolution during TOF analyses) with the use of 6-aza-2-thiothymine as matrix. The MALDI technique is widely credited for acceptance of significant sample contamination by inorganic salts; such tolerance (which is arguably overstated even for the most amenable of analytes) emphatically does not apply to the analysis of oligonucleotides. Substitution of alkali metal cations by ammonium ions is highly beneficial to the achievement of intense signals (G12) [paralleling observations with analyses using liquid SIMS (G1) and electrospray ionization (G17)]. Currie and Yates (G18) used a matrix of 2,5-dihydroxybenzoic acid mixed with ammonium acetate; they suggest, however, that the effect of the ammonium ions may extend beyond simple displacement of residual metal cations and may include the promotion of gas-phase ion molecule interactions favoring oligonucleotide ion formation and the enhancement of gas-phase ion cooling. Several strategies have been adopted for the removal of salts on the MALDI sample probe. Lubman and co-workers (G19) used a Nafion substrate in conjunction with 3-hydroxypicolinic acid matrix. Nordhoff et al. (G12) mixed cation exchange polymer beads with the matrix. Extensive fragmentation of oligonucleotide ions produced by MALDI is frequently observed; this may contribute to reduced mass resolution, particularly when a linear TOF analyzer is used, and reduced sensitivity if a reflectron is incorporated. The propensity to fragment is markedly greater for oligodeoxynucleotides (G10, G20). Wu et al. (G15) compared signal intensities in the positive and negative ion mode during linear and reflectron TOF analyses of single-stranded DNA (10-89-mers). Linear TOF analyses indicated similar yields of ions of both polarities, using 3-hydroxypicolinic acid as matrix (as noted above), but the lesser intensity of positive ion spectra recorded using the reflectron analyzer indicated a greater tendency to fragment than for negative ions derived from the same oligonucleotide (G15). Fragmentation occurring in the flight tube between the ion source and reflectron can be put to analytical use in postsource decay experiments employing stepped reflectron voltages or a curved field reflectron (see section C). Nordhoff et al. (G6) have noted a greater degree of “prompt” (in-source) fragmentation of negative oligonucleotide ions following IR as compared with UV MALDI, yielding spectra with extensive sequence information. These authors suggest mechanistic distinctions between prompt, charge-remote fragmentations and metastable, charge-driven decompositons occurring after ejection from the ion source (G6). The extent of prompt fragmentation is increased if a delay is incorporated between the ionizing laser pulse and the extraction potential pulse, increasing the abundance of sequence-specific fragments (G21). Two groups have independently noted the enhanced stability of nucleic acid ions incorporating 7-deazapurine bases (G22, G23), with implications for the use of MALDI MS in DNA-sequencing experiments. Schneider and Chait (G22) explicitly suggest the potential use of 7-deazapurine analogues in the Sanger dideoxy sequencing method for DNA with MALDI readout of the sequence. An example of successful “ladder” sequencing of a small (12mer) synthetic oligodeoxynucleotide has been presented by Pieles et al. (G24). The products of a series of partial digestions, 632R
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employing either 3′- or 5′-exonucleases with different digestion times, were combined and analyzed by MALDI. The preferred matrix was 2,4,6-dihydroxyacetophenone with di- and triammonium salts of organic or inorganic acids to suppress metal cationization. Exonuclease digestion of a larger RNA has been reported by Kirpekar et al. (G10). The ladder sequencing approach is likely to find increasing application, paralleling similar developments in the analysis of peptides (G25, G26). Williams (G27) has reviewed his group’s work on the laser ablation and ionization of DNA from thin frozen films mounted on oxidized copper substrates. Collection of ablated material has indicated successful ablation of intact DNA as large as ∼400 kDa in mass. Ions of single-stranded DNA up to ∼18.5 kDa have been detected using time-of-flight MS. The potential advantage of this approach appears to be a minimization of ion fragmentation, but this is presently achieved at the expense of substantially less experimental convenience than is associated with the MALDI experiment. Significant refinements to the application of electrospray ionization to the analysis of oligonucleotides have also been reported in the last two years. As with MALDI analyses, the removal of metal cations is critical to success. Following the early report of Stults and Marsters (G17) on the beneficial effect of sample treatment to displace metal cations by ammonium ions, this general approach has been commonly adopted. Limbach et al. (G28) have described a strategy in which precipitation in the presence of ammonium acetate is followed by the addition of chelating agents such as trans-1,2-diaminocyclohexane-N,N,N′,N′tetraacetic acid to remove residual traces of divalent metal ions and triethylamine to remove monovalent cations. This combined strategy sufficiently improved spectral quality that analyses of RNAs to ∼40 kDa in mass yielded mass measurement accuracy of better than 0.01% (G28). Addition of triethylamine to the electrospray solvent has been noted to be beneficial (G29, G30). Grieg and Griffey (G31) observed, however, that addition of either triethylamine or piperidine to the electrospray solvent could reduce oligonucleotide ion abundances as well as effectively suppress metal cationized species. Co-addition of imidazole effected analyte signal enhancement without loss of the benefit of reduction of metal cationized species. In addition, there was a slight shift to lower charge states (G31). Smith and co-workers (G32, G33) have focused on the issue of charge-state reduction during electrospray analyses of oligonucleotides, with the potential benefits of simplification of the spectra obtained and concomitant increases in sensitivity. A cocktail including imidazole, piperidine, and acetic acid in 80% acetonitrile reduced charge states and suppressed formation of metal cationized species (G33). The benefit to sensitivity of solvent incorporating a high percentage of organic constituent substantiated the earlier findings of Bleicher and Bayer (G29). Tong et al. (G34) have compared charge state distributions under similar experimental conditions for d(pA)6, d(pC)6, d(G)6, and d(pT)6; an apparent correlation was observed between charge state and the basicity of the nucleoside bases (pKa), suggesting that charge-state reduction may result from internal charge neutralization attributable to gas-phase protonation of the nucleoside base. Approaches to oligonucleotide sequencing using electrospray MS have included exonuclease degradation and analysis of the resultant fragments (G35, G36), a strategy identical to that
described above in applications of MALDI. Adducts of styrene oxide with DNA have been recognized by combined capillary electrophoresis/electrospray MS of the products of nonspecific enzymatic digestion yielding a mixture of dimeric to hexameric oligonucleotide products (G37). Promotion of fragmentation in the electrospray interface or in the collision region of a true tandem MS instrument is suggested as a means of defining precise sites of modification (G37). Systematic investigations of the fragmentations of multiply charged oligonucleotide anions generated by electrospray have been undertaken by McLuckey and colleagues (G38-G42). Studies on the fragmentations of singly charged oligonucleotides (G43, G44) and of all possible heterodinucleotides (G45) provide useful background. McLuckey et al. (G38) reported the principal fragmentation pathways observed during tandem MS analyses using a Paul ion trap and suggested a common nomenclature. The effect of base modification on the collision-induced fragmentation of multiply charged oligonucleotide anions has been studied by Barry et al. (G46). Crain et al. (G47) have detailed the CAD of [M - 4H]4- ions derived from the octamer d(CGAGCTCG), an example chosen because of prior reports of tandem MS analyses of the same or similar sequences using Paul ion trap (G38) and ICR (G48) instruments. On the basis of this limited evidence, the yield of sequence specific information from CAD/ tandem MS analyses using a triple quadrupole instrument may exceed that obtained from equivalent experiments on the alternative instrument types, presumably reflecting differences in the time scales of the decomposition experiments. Much more extensive data are required, however, to assess the security of this generalization. More generally, a significant service would be provided by the preparation of a detailed critical review of the fragmentation of oligonucleotide ions of both polarities and including singly and multiply charged ions, observed under various experimental conditions. Such a treatment is well beyond the scope of the present review. The capability of the Paul trap to achieve simultaneous trapping of both cations and anions has been exploited by Herron et al. (G49) in the study of proton transfer reactions between protonated pyridine and multiply charged oligonucleotide anions. Interestingly, little fragmentation was observed to result from proton transfer, despite the exothermicity of reaction. Potential advantages attach to the use of FT-ICR instruments for the analysis of oligonucleotides. The molecular mass of a 100mer single-stranded DNA has been determined using electrospray ionization and FT-ICR (G50); the substantial experimental difficulty of identifying the monoisotopic peak in an isotopic distribution resolved in such analyses is addressed by comparison of the observed distribution with that calculated for an “average” oligonucleotide of the same average molecular mass (G51). The facility for determination of molecular masses with an accuracy and precision not achieved by other instrument types limits the number of possible base compositions corresponding to the determined mass (G48, G50). The resolution achieved using FTICR may also benefit the identification of dissociation fragments. For example, electrospray FT-ICR analysis of d(CGAGCTCG) with promotion of decomposition in the interface region yielded resolved fragment ions of measured relative mass 427.019 and 427.097, attributed to (w1 + H + HPO3) (calculated mass 427.019) and (w1 + H + C5H4O) (calculated mass 427.078) (G48). Computer algorithms based on pattern recognition techniques
have been developed to facilitate assignment of charge states to resolved isotopic clusters of fragment ions (G52). In the studies of McLafferty and co-workers (G48), sequence information is generated both by induced decomposition in the electrospray interface (by application of an appropriate potential between the nozzle and skimmer components of the interface) and by collisional activation of selected trapped ions in the ICR cell. Both techniques, of course, rely on activation of precursor ions by collision with gas atoms or molecules; differences between the observed fragmentation patterns may be associated with the prior selection of a single precursor ions species (in the true tandem MS experiment), differences in the energetics and multiplicities of ion/neutral collisions, and the time scales of the experiments. Little and McLafferty (G53) have also argued for the complementary value of induced decomposition in the electrospray interface and infrared multiphoton dissociation of trapped ions; sequence data for a 50-mer DNA have been reported (G53). In studies of coliphage T4 DNA, Smith and co-workers (G54) have demonstrated the experimental realization of the predicted (G55) capability of FT-ICR to detect single (individual) ions of very high mass and charge number. Direct determination of charge number (G56) (albeit with a precision presently estimated as (10%) enables conversion of observed m/z values to mass estimates. The data establish the capability, using electrospray ionization, of producing stable gas-phase DNA ions of approximate mass 110 MDa, with charge numbers in excess of 30 000 (G54). Electrospray ionization has been widely used in the detection of noncovalently bound dimeric ions, the observation of which has been frequently taken to reflect condensed-phase chemistry. [In the last review in this series (G57), however, we urged caution in interpreting both qualitative and quantitative data from the observation of noncovalently bound species. We suggest that the warning remains apposite and the discussion that follows should be viewed in this light.] The physiological importance of basepaired oligonucleotide hybridization has prompted several laboratories to investigate the formation of specific oligonucleotide dimeric ions following electrospray ionization. Ganem et al. (G58) described the detection of dimeric species of self-complementary octanucleotides using electrospray MS. A weak signal corresponding to heterodimeric dT8 and dA8 was observed during analysis of a mxture of these complementary sequences; tandem MS with CAD of the dimeric species substantiated noncovalent interaction. Smith and co-workers (G59) reported the detection by electrospray ionization of a duplex of complementary 20-mers; successful analysis was promoted by careful attention to “gentle” interface conditions. The use of an extended m/z-range quadrupole analyzer was helpful in that the intact duplex was observed at significant abundance only in relatively low charge states. Smith et al. (G60) also reported the detection of an oligonucleotide quadruplex of d(CGCG4GCG) when electrospray analyses were performed using sample solutions containing monoatomic metal cations known to stabilize quadruplex formation. A recent report from Greig et al. (G61) suggests the benefit of using low flow rate electrospray (see section C) to enhance the detection of a DNA duplex relative to the constituent monomers. Bayer et al. (G62) investigated the detection by electrospray MS of duplexes formed between a single strand and complementary sequences; the signal intensity corresponding to a duplex increased with the length of the complementary strand, suggesting a correlation with duplex stability. The gas-phase stability of Analytical Chemistry, Vol. 68, No. 12, June 15, 1996
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oligonucleotide duplex ions under the conditions of analysis in the Paul ion trap has been investigated by McLuckey and co-workers (G63); the data suggest a 10-mer/10-mer duplex ion lifetime of at least 100 ms in the presence of a thermalizing bath gas at a pressure of 1 mTorr. Extended trapping lifetimes allow the use of ion excitation techniques to promote and determine precursor ion fragmentation. The potential for nonspecific oligonucleotide dimer formation has been noted by Ding and Anderegg (G64); they observed that dimer formation was concentration dependent and was nearly always observed when concentrations exceeded 100 µM, regardless of complementarity. Nevertheless, careful comparison of the expected and observed relative abundances of homo- and heterodimeric ions during electrospray analyses of oligonucleotide mixtures suggested a degree of specificity for the complementary association. A correlation was suggested between specific dimerization and the number of hydrogen bonds within each pair of oligonucleotide strands (G64). Lecchi and Pannell (G65) have reported evidence for the detection of an oligonucleotide duplex using MALDI. A duplex composed of a 16-mer and complementary 12-mer was investigated to permit distinction between homo- and heterodimers. Elevated temperatures and acidic pH were avoided during sample preparation and 6-aza-2-thiothymine with ammonium citrate was used as the MALDI matrix. The mass spectrum included an abundant ion corresponding to the heterodimer (duplex). Concomitant detection of the monomers was attributed to their presence in the original sample (rather than arising from sample preparation or analysis) because their relative abundance was diminished by prior treatment of the sample with a nuclease which preferentially attacks the single strands (G65). MALDI has also been used to analyze DNA duplexes in which one strand is biotinylated and immobilized to streptavidin-coated magnetic beads which are applied to the target (G66). Desorption/ionization yields only the nonimmobilized strand. Suggested applications of this approach include fast DNA sequence analysis by extension of classical Sanger methodology (G66). ENVIRONMENTAL RESEARCH AND TOXICOLOGY Mass spectrometry plays a crucial role in environmental chemistry, in view of its effectiveness as an on-line chromatographic detector of excellent (though not always optimum) sensitivity, its universality yet with potential for selectivity and specificity, and its amenability to quantitative analysis over a wide dynamic range. These properties account for the importance of mass spectrometry in the analytical methods prescribed by the Environmental Protection Agency for analysis of target analytes in environmental matrices. Development of methods of this kind continues as the list of anthropogenic pollutants of concern for environmental and human health continues to grow. A more recent trend, however, directs its attention to the modes of action of these pollutants in harming living organisms, including human beings. This section will focus primarily on the latter aspect, which accounts for the title chosen for the section. Pollutants in the Environment. Hites (H1) has written an excellent short article which puts into perspective some current issues in the interplay between development of analytical techniques and their use in following the fate of pollutants in the environment. A good example of this interplay is provided by work on chlorinated organic pollutants. Negative chemical ioniza634R
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tion (NCI) provides increased sensitivity for electronegative analytes by up to 2-3 orders of magnitude, compared with that provided by electron impact ionization or positive ion CI (PCI), but in the past has been notoriously difficult to control sufficiently to provide reproducible data (H2, H3). Clearly this situation had to be brought under control for the exacting experimental work involved determining quantitative data on environmental samples, and a series of interlaboratory comparisons and fundamental investigations (H4-H6) has identified those experimental parameters which must be carefully controlled in NCI mass spectrometry. Some of the global environmental conclusions, which have been made possible by acquiring reliable NCI data in this way, are described by Hites and his collaborators (H7-H9). More recently, a new development in NCI has been described by Larame´e and Deinzer (H10-H12). In order to avoid many of the difficulties associated with the moderating gas used to thermalize the electrons in a conventional NCI source, these workers have developed an electron monochromator which can deliver useful intensities of electrons with tunable energies in the range 0.03-30 eV with an energy spread of 0.36 eV. The advantages of this approach include the possibility of tuning the electron energy to match the resonance anion states of the analytes, thus providing an additional degree of experimental discrimination among isomers, for example. These negative ion resonance capture states range up to 7 eV in extreme cases, but are mostly well below this upper limit, so that competition from EI to produce positive ions is not a factor. Because no CI plasma is present, complications from reactions of analyte molecules with the radicals and ions present in such plasmas, which can complicate conventional NCI (H1-H6), are avoided, and the ion source is much less susceptible to contamination. This device, based on an electron monochromator design due to Fenzlaff and Illenberger (H13), has been shown to perform well for several classes of electronegative compounds of environmental importance, providing three-dimensional mass spectral information (ion intensity as a function of m/z and of electron energy) on the GC time scale. The results reported thus far from this ambitious development (H10-H12) are encouraging, even though the sensitivity is not yet sufficient to provide a routine technique for real-world samples at the levels currently handled by conventional NCI with appropriate precautions (H4-H6). The design improvements underway (H12) focus on increasing the intensity of the electron beam delivered to the ionization chamber, and investigation of low gas pressures to stabilize molecular anions against autodetachment without too great a deterioration of the electron energy resolution. While NCI is extremely important in applications of mass spectrometry to environmental analysis, it provides only one of many tools for this purpose. Miles et al. (H14) have published a short paper demonstrating the interplay of several techniques, including NCI, PCI, EI, selected ion monitoring at high resolution (104, 10% valley definition), and MS/MS, in the analysis of environmental pollutants amenable to GC separation. The limits on those compounds amenable to GC/MS analysis have been extended to less volatile but still thermally stable analytes by interfacing a mass spectrometer to high temperature capillary GC columns operating at up to 450 °C or more (H15). Many compounds of environmental concern are simply too involatile and/or thermally unstable to be analyzed by GC/MS, and suitable derivatization is not always possible. Some of the
so-called “nonpersistent” pesticides fall into this category, as do many dyestuffs. Of the approximately 150 million pounds of azo dyes produced in the United States each year, about 15% are discharged into waste waters (H16). Many of these dyes and their metabolites are believed to be carcinogenic (H16, H17). Mass spectrometric analysis of these highly polar and thermally labile compounds requires use of condensed-phase ionization techniques, and preliminary investigations have delineated the fragmentation mechanisms of ions (frequently sodiated in the case of sulfonated azo dyes) formed by a variety of techniques including FAB, thermospray, and plasma desorption, as summarized by Borgerding and Hites (H18) in a recent paper on the FAB tandem mass spectra of these compounds. Methodologies for environmental monitoring will require mass spectrometry coupled to highresolution separatory techniques, and to this end coupling of HPLC with APCI (H19) and with electrospray (H20) with MS/ MS analysis, and with thermospray (H21) has been described, while the potential of CE/MS/MS for analysis of sulfonated azo dyes has also been explored (H22, H23). The limited sensitivity of electrospray ionization for sulfonated dyes is due, at least in part, to the adduction of cations such as sodium and potassium, and the effect of additions of amine bases on improved ionization efficiencies has been reported by Ballantine et al. (H24), following the earlier findings of Greig and Griffey (H25) for nondesalted oligonucleotides. The combination of electrochromatography with electrospray ionization has been investigated as an analytical method for nonionic textile dyes (H26). Despite all of these efforts, it appears that no analysis of a real-world environmental sample for azo dyes using mass spectrometry has been reported, although analyses of spiked environmental matrices are described in some of the literature cited. Indeed, the only report of analysis of a textile mill waste water in the refereed literature appears to be that due to Camp and Sturrock (H27), who used HPLC with a swept-potential electrochemical detector rather than mass spectrometry. Other chemical classes of dyestuffs, such as triarylmethine and xanthene dyes, are used in food and cosmetics. As pointed out by Borgerding and Hites (H28), despite their direct consumption by humans and their known genotoxic properties (H29), even less attention has been devoted to the analysis and environmental behavior of these compounds than of the azo dyes. Most analytical methods use HPLC with UV or fluorescence detection. Until recently the only published study of the mass spectrometry of food dyes was that of Harada et al. (H30), who investigated their reduction under FAB ionization conditions, and demonstrated poor sensitivity. More recently (H28) HPLC with continuous-flow FAB was used to analyze waste water samples containing a range of food dyes. Only a synthetic precursor of one of the dyes could be detected by mass spectrometry, so that UV detection, with considerable preanalysis cleanup, had to be used for the other analytes. This dearth of reports of analyses of potentially dangerous pollutants emphasizes the difficulties involved and provides both a challenge and an opportunity for the mass spectrometry community. The discussion thus far has emphasized development of new techniques and methodologies for environmental analysis. Exploitation of proven mass spectrometry techniques to investigate larger questions concerning distribution and fate of pollutants in the environment are too numerous for complete coverage here. These include the work on organochlorines due to Hites et al. (H7-H9), mentioned above. Another example is provided by the
work of Simonich and Hites (H31, H32) on the role of vegetation in removing polycyclic aromatic hydrocarbons from the atmosphere. An excellent review article by Charles and Feinberg (H33) describes the contributions of mass spectrometry to three problems in environmental science, viz., chlorination reactions of humic and fulvic materials in water, atmospheric photooxidation of hydrocarbons (both natural and anthropogenic), and atmospheric sources, fate, and transport of organics in aerosols. Charles and collaborators have themselves contributed to some of these topics, e.g., identification of a large range of compounds [including the potent mutagen MX, or 3-chloro-4-(dichloromethyl)5-hydroxy-2(5H)-furanone, and related compounds] in extracts of aqueous fulvic acid treated with monochloramine (H34). It is revealing that more than 50% of the total organic halogen in chlorinated humic samples and in chlorinated water supplies remain unidentified (H33), a situation similar to that for organochlorines in the lipid fraction (cyclohexane extractable) in exposed fish and sediments (H35, H36). Charles et al. (H37) have developed a derivatization procedure, which enables identification of carbonyl compounds in environmental samples by GC/ MS, and have also made a significant contribution to mass spectrometric techniques for analysis of polychlorinated dibenzop-dioxins and dibenzofurans via a systematic investigation of MS/ MS optimum conditions and a comparison of the merits of this approach relative to high-resolution selected ion monitoring (H38-H40). The foregoing represents only a small selection of recent work in which mass spectrometry has contributed to environmental science and reflects the reviewers’ bias toward those publications in which mass spectrometry played a crucial role. With regard to development of techniques and methodologies, other examples with implications for environmental analysis can be found elsewhere in this review; e.g., the recent revival of membrane inlet mass spectrometry is described in section C. An extensive review of applications of LC/MS techniques to environmental analysis (H41) covers pesticides and herbicides, polycyclic aromatic hydrocarbons, organometallics, dyes, and other miscellaneous compounds. Direct Detection of Genotoxicity. The following discussion of applications of mass spectrometry to characterization of chemically altered DNA and RNA inevitably duplicates some of section G, which deals with more general aspects of the mass spectrometry of DNA and oligodeoxynucleotides and nucleosides. Miller (H42) has described historical discoveries that led to modern research on chemical carcinogenesis, including early observations of scrotal skin cancer in chimney sweeps, demonstration of induction of skin cancer by coal tar, and isolation of benzo[a]pyrene (BaP) as an active agent in coal tar followed by more detailed studies of initiation and promotion stages of skin cancer by BaP. A brief account of these same historical foundations has been given by Maccubbin (H43). The first indications that chemical carcinogenesis might be related to covalent binding of chemicals to DNA were published in the late 1950s and early 1960s (H44-H47), and since then numerous DNA adducts with a variety of chemicals have been described (H48). Those chemical carcinogens which cause damage to DNA are called genotoxic, while those which do not directly cause damage are termed nongenotoxic, although the distinction becomes less clear in cases of carcinogenic compounds which damge DNA by stimulation of radical production. Many genotoxic carcinogens Analytical Chemistry, Vol. 68, No. 12, June 15, 1996
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and/or their metabolites are electrophilic and react with nucleophilic sites in the DNA bases and with the phosphate oxygen to form the covalent adducts. Such electrophiles react also with other nucleophilic constituents of the cell, including appropriate amino acid side chains and both N- and C-terminus groups of proteins, as well as with glutathione to form S-alkylglutathione detoxification products (H49, H50). Formation of a DNA adduct is believed to be a necessary “initiation” event in chemical carcinogenesis by genotoxic agents, but is not in itself sufficient for a tumor to develop. The subsequent “promotion” and “progression” stages may also be associated with exogenous chemicals, not necessarily the initiating genotoxic agent. Mass spectrometry played no role in any of this early work (H42-H46) on genotoxin-DNA adduct formation. The sensitivity of the technique was simply inadequate to deal with the low extent of adduct formation in exposed cells, as low as one modification in 108 nucleotides. In the case of human studies, DNA samples may be available only in quantities less than 100 µg, which implies that the sample contains only 1 pg or so of modified nucleotide. Use of radiolabeled carcinogens provides high sensitivity but is of limited applicability to human studies and, in itself, provides no information on adduct structures. Other methods of investigation not involving mass spectrometry have been briefly reviewed by Maccubbin (H43), who also conveniently tabulates their sensitivities in terms of the minimum ratio of adducted to native nucleotides that can be detected and of the quantities of DNA required for analysis. Thus, HPLC with fluorescence detection requires about 100 µg of DNA and, in suitable cases, can detect 1 adducted nucleotide in 107. When suitable antibodies are available, immunochemical approaches can also detect 1 adducted nucleotide in 107, but using only 1 mg or so of DNA (H43). The most widely used method of detection for modified nucleotides in DNA is the 32P-postlabeling technique of Randerath et al. (H51, H52), in which DNA is enzymatically hydrolyzed to the 3′-nucleotides, and then converted to 32P-labeled nucleoside-3′,5′-diphosphates by treatment with γ-[32P]ATP and a nucleotide kinase. Following multidimensional chromatography on cellulose TLC plates to separate normal from adducted nucleotides, autoradiography of the TLC plate permits detection and quantitation of the adducts at levels as low as 1 in 109 using 1-10 µg of DNA (H51, H52). If authentic standards are available it is possible to match Rf values and make tentative identifications, but the method suffers from lack of structural specificity and limited resolution [Maccubbin (H43) reproduces examples of such autoradiograms]. There is a real need for mass spectrometric methods to provide molecular weight and structural information for these adducts, at levels comparable to those detectable by the Randerath procedure. The structures of DNA adducts can provide important clues regarding the carcinogen metabolism processes responsible for the DNA modification, and this is indeed a challenge for mass spectrometry. Application of mass spectrometry to characterization of nucleosides dates back to early work of Biemann and McCloskey (H53). This early work on EI fragmentations of nucleosides was later reviewed by McCloskey (H54). Since that time progress in mass spectrometric characterization of DNA and RNA has followed developments in mass spectrometric methodology. The development of GC/MS methods for positive ion analysis and quantification of the DNA bases as their trimethylsilyl derivatives has been described in a recent review article (H55). The use of GC/MS 636R
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with NCI of free bases derivatized by the attachment of pentafluorobenzyl groups to amine nitrogens or hydroxy oxygens was introduced by Vouros and Geise et al. (H56-H58) and can detect 1 modified base in 105-106 using 1 µg or less of DNA (H43), providing molecular weight but no structural information. Current progress in the application of mass spectrometry to DNA and its constituents has benefited from continuing development of ionization techniques applicable to highly polar molecules. Recent general reviews include those by Schram (H59), by Crain (H60), by Vigny and Viari (H61), and by McCloskey (H62). A review of the essential preliminary steps of preparation and enzymatic hydrolysis of DNA and RNA for mass spectrometry has been written by Crain (H63). One of the most significant contributions of mass spectrometry to solving real problems in nucleic acid chemistry and biochemistry has involved characterization of natural modifications, primarily in RNA as described by McCloskey (H64) and by Crain and McCloskey (H65), but also in DNA (H66, H67). The present section, however, is restricted to application of the mass spectrometric approaches, described in detail elsewhere (H59-H67), to detection, characterization, and (where possible) quantification of DNA bases modified by adduction to xenobiotic chemicals. Some excellent recent reviews on this subject are due to Chiarelli and Lay (H68), McCloskey and Crain (H69), and Farmer and Sweetman (H70). An important class of chemical modifications to DNA arises from oxidative damage. Aerobic organisms must balance the positive effects of oxygen against the generation of reactive oxygen species which have been estimated (H71) to give rise to 1011 radicals per cell per day. Defense mechanisms include radical scavengers but various stresses, including those imposed by some xenobiotic substances, can lead to oxidative damage to DNA (as well as to lipids and proteins) as an ongoing process. This oxidative damage to DNA is countered by the evolution of enzymes devoted to DNA repair, but there is strong evidence (H72) linking such damage to spontaneous mutagenesis leading to cancer and aging. Much of this work on oxidative modifications of DNA has involved analysis using HPLC with electrochemical detection (H73), but the levels of modified bases (1 modified base in 105-107) are such that mass spectrometric methods are feasible at least for the higher modification levels. Dizdaroglu (H55, H74, H75) has developed protocols for positive-ion GC/MS analysis of nucleotide bases following strong acid hydrolysis of DNA (150 °C for 90 min) and trimethylsilylation, but the levels of oxidized bases thus detected are generally much higher than those reported using HPLC analysis (H73). This discrepancy has been attributed tentatively (H73) to artifactual oxidation by the strong hydrolysis conditions or possibly during the derivatization (H74, H75). More recently, Wagner et al. (H76, H77) have developed a methodology involving either hydrolysis under much more mild chemical conditions followed by separation from nonmodified bases to minimize artifactual production of oxidized bases, or selective enzymatic excision of modified pyrimidines (nonmodified bases remaining within the DNA). This procedure (H76, H77) then calls for fluorobenzylation and analysis by GC/MS using NCI (H54-H56), thus providing excellent instrumental sensitivity together with the specificity provided by mass spectrometric determination of molecular mass. Provision of 13C- and 15N-labeled internal standards (H77) for some oxidized bases should also permit reliable quantitation. A somewhat similar procedure has
been reported by Teixeira et al. (H78) to permit quantitation of oxidized bases at a level of 20 per 106 using only 30 µg of DNA, and with variability of less than 5 and 8% for within-run and between-run precision, respectively. The use of GC/MS with NCI, for characterization of other DNA adducts with endogenous compounds, e.g., malondialdehyde, an end product of lipid peroxidation, has been described by Blair et al. (H79, H80). In disease-free human liver, levels of 1 target adduct per 106 bases could be detected (H80), comparable to those of bases subject to direct oxidative attack and to the highest levels of DNA adducts found for exogenous genotoxic compounds. Although NCI mass spectrometry will undoubtedly continue to be a useful tool in such research, avoidance of the derivatization step should in principle provide a more reliable analytical method, and to this end the potential of the more modern ionization techniques for polar analytes has been investigated. Early work of Straub et al. (H81) on field desorption (FD) ionization of DNA adducts of the activated metabolite of BaP (7,8-dihydroxy-9,10epoxy-7,8,9,10-tetrahydro-BaP) showed disappointing sensitivity compared with that observed in the same work for permethyl or persilyl derivatives by high-resolution EI with accurate mass measurement. Nonetheless FD studies provided early confirmation of structures of DNA adducts with several exogenous compounds. The FD work up to about 1980, including CID studies of BaP adducts, was summarized in two reviews by Straub and Burlingame (H82, H83). Because of the experimental difficulties with FD, since 1981 this work has been largely replaced by fast atom bombardment (FAB) and other ionization techniques. A large number of structural studies of DNA nucleosides covalently modified by genotoxic compounds, using FAB ionization, has been published. Many of these are described in the excellent review by Chiarelli and Lay (H68), and selected examples are reviewed by Farmer and Sweetman (H70). While these structural studies on DNA adducts formed in vitro are invaluable, particularly those employing MS/MS, the sensitivity of FAB was disappointing relative to the anticipated requirements for in vivo studies. Annan et al. (H84) showed that FAB sensitivity in analysis of arylaminedeoxynucleoside adducts was greatly enhanced by trimethylsilylation, a result confirmed by other groups but disappointing in that the hoped-for advantage of avoiding derivatization is thus discarded. Wolf et al. (H85) exploited the increased sensitivity of continuous-flow FAB (CF-FAB) for DNA adduct analysis and have also used LC/MS with CF-FAB to analyze the reaction products of N-acetoxy-N-acetyl-2-aminofluorene (AAF) with calf thymus DNA (H86). In the latter example, full-scan and MS/ MS data provided useful structural information on the adducts for low-nanogram (low-picomole) quantities, while the instrumental limit of detection using HPLC with multiple-reaction monitoring was about 25 pg (50 fmol) indicating the potential for screening of deoxynucleoside adducts with AAF at a level of about 1 in 106 from about 1 mg of DNA. While an impressive accomplishment, this level of sensitivity (H86) is still some 2 orders of magnitude below the ultimate target described above. Similar continuousflow FAB sensitivities for PAH adducts of deoxynucleosides have been reported by Wellemans et al. (H87). The use of thermospray to facilitate LC/MS analysis of hydrolysates of DNA and RNA has been particularly useful in the hands of McCloskey et al. in the discovery of previously unknown nucleosides from tRNA (H64, H65, H88). However, low sensitivity is undoubtedly the cause of the scarcity of reports on the use of thermospray to
characterize deoxynucleoside adducts. Korfmacher et al. (H89) reported early thermospray LC/MS data for some arylamine adducts, and Van der Poll et al. (H90) used the same technique in conjunction with FAB mass spectrometry and also NMR to characterize large quantities of adducts formed by in vitro reactions. Thermospray LC/MS has been used (H91) to characterize µg quantities of in vitro DNA adducts with anticancer drugs thought to act via their ability to bind to DNA forming monoand dialkylated products (H92). The advent of electrospray ionization and MALDI over the last few years has naturally directed efforts to their use in determining DNA adducts. Crain and McCloskey (H65) have described recent applications of electrospray in characterizing naturally modified nucleosides and oligonucleotides from RNA. The application of these two ionization techniques to detection and hopefully quantitation of covalently modifed DNA bases depends upon whether or not sequence information is desired. If the intent is to complement the 32P-postlabeling procedure (H51, H52), by characterizing the mononucleosides obtained by the total hydrolysis of the modified DNA, or possibly the bases themselves, LC/ MS/MS using electrospray ionization holds some promise. Quilliam (H93) has shown that target analysis using isocratic LC/ MS with SIR could detect levels of modified base corresponding to a base modification level of about 1 in 106, an instrumental detection limit subject to improvement using gradient elution (larger injection volumes) and other techniques. More recently Chaudhary et al. (H94) investigated the potential of LC/MS/MS with electrospray applied to endogenous nucleoside adducts, and found an instrumental detection limit for an authentic standard of 10 pg (signal/noise > 15) and a linear response over the range 10 pg to 10 ng (this work used gradient elution). The general conclusion of this work (H94) was that LC/MS/MS with MRM is about 1 order of magnitude less sensitive than GC/MS with NCI, but that the ease of use of the electrospray technique makes it an attractive alternative. This generally optimistic conclusion was also reached in the work of Rindgen et al. (H95), who investigated LC/MS/MS techniques (incorporating electrospray) for detection of DNA adducts of a heterocyclic aromatic amine. Gradient elution with MRM was again used, and a detection limit of 38 pg for the pure standard was reported, with linear response up to 1 ng. For qualitative (e.g., constant neutral loss scanning) analysis of unknown samples, the sensitivity was 2-3 orders of magnitude less (H95). Exploitation of the advantages offered by MALDI in analysis of modified mononucleosides and/or bases currently requires preseparation and collection of the modified components from the much larger quantity of unmodified bases, rather than on-line LC/MS coupling as is possible with electrospray. Fraction collection of this type has been significantly advanced by the work of Monnig et al. (H96, H97) in developing an automated fraction collector for capillary electrophoresis in which the fractions are deposited directly on to MALDI probes. Thus far this technique has been demonstrated only for proteins and peptides, however. Lay et al. (H98) have demonstrated lowfemtomole detection of nucleoside adducts with 4-aminobiphenyl, using MALDI/TOF analysis. Stemmler et al. (H99) have investigated the potential of the MALDI/FT-ICR combination to give detailed information (accurate mass measurement and MS/MS) for PAH diol epoxide adducts of both mononucleosides and mononucleotides, using sample sizes in the range 10-40 ng. George et al. (H100) investigated rational optimization of matrix Analytical Chemistry, Vol. 68, No. 12, June 15, 1996
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selection for MALDI-TOF analysis of PAH adducts of the free DNA bases, and obtained detection limits in the low-femtomole range. Finally, it is interesting that a much more mature technology, that of desorption chemical ionization, in combination with MS/MS techniques, has been shown by Wood et al. (H101) to provide detection and quantitation of methyl-mononucleosides from a DNA hydrolysate, to the subpicomole level. If the objective is to acquire information about the location of the modified base in the DNA, i.e., sequence information on oligonucleotides, additional difficulties arise in the application of mass spectrometry. One of these additional problems for FAB and electrospray ionization concerns the affinity of the phosphate groups for cations such as sodium and potassium, and various methods have been described for desalting natural nucleic acids and oligonucleotides (H87, H102-H104). Alternatively, addition of organic solvents (H105) or of organic amines (H25) to the analyte solution can significantly lower the extent of cation adduction and increase electrospray sensitivity for oligonucleotides without desalting. Limbach et al. (H106) have shown how the problem of metal cation adduction to nucleotides can be overcome through a combination of minimal pretreatment and addition of chelating agents to remove divalent metal ions and of triethylamine to displace monovalent cations from the nucleotides (RNA in the examples studied (H106)). In this way it was possible to obtain mass measurements to within 0.01% using a quadrupole analyzer, without complex cleanup procedures. The effect of electrospray source parameters on the mass spectra of mononucleotides has been investigated (H107), and the effect of basicity of the naturally occurring DNA bases on the charge state distributions in electrospray mass spectra has been studied (H108). McLuckey et al. (H109-H111) have demonstrated the ability to sequence short oligonucleotides using MS/MS/MS techniques on multiply charged ions in an ion trap, and Little et al. (H103) have reported sequencing oligomers up to 25 bases in length using FT-ICR techniques. However, none of these MS/MS studies involved oligonucleotides incorporating modified bases. Recently Barry et al. (H112) investigated the MS/MS spectra and fragmentation mechanisms of triply deprotonated synthetic oligonucleotides CACGXG, where X denotes a modified pyrimidine base. Traditional sequencing methodologies frequently fail in the characterization of modified oligonucleotides. As emphasized by Barry et al. (H112), the Sanger method uses DNA polymerases which may fail to recognize the modified template and prematurely halt the extension of the complementary chain at the position of the modification or may introduce a randomly chosen deoxynucleotide, while Maxam-Gilbert reagents often fail to cleave the phosphate backbone at the location of the modified base. Such failures of traditional sequencing methods are even more probable when the backbone of the oligonucleotide has been modified, e.g., to methylphosphonate. At present, the main role of mass spectrometry in oligonucleotide sequencing is concerned with such modifications. Successful approaches to mass spectrometric sequencing have included the work of Pieles et al. (H113), who used a two-enzyme procedure to degrade the oligonucleotide in a stepwise fashion, yielding a ladder of successive oligomers which were successfully analyzed by MALDI/TOF to give the sequence of a 12-mer. Somewhat similar approaches using electrospray ionization to characterize the ladder were reported by Glover et al. (H114), who used HPLC to separate the reaction products with off-line mass spectrometric analysis using an imidazole buffer 638R
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with 70% acetonitrile to increase ionization efficiency and reduce sodium adducts (H25, H106). Stemmler et al. (H115) have investigated the use of MALDI with FT-ICR in characterizing modified oligonucleotides at the low-picomole level and found that choice of matrix helped determine the degree of fragmentation observed. It was possible (H115) to determine the sequence, including the position and identity of the modified base, for the 4-mers and 6-mers investigated, but in the case of the 11-mer the sequence at the 5′ end could not be determined. A protocol for mass measurement (to within 0.005%) of natural, modified, and antisense oligodeoxynucleotides, using electrospray ionization, has been described by Deroussent et al. (H116). An excellent review of work up to 1993 on all aspects of PAH-DNA adducts, particularly the use of mass spectrometry in their characterization and measurement, has been written by Giese and Vouros (H117). It is clear that a great deal of creative effort has been devoted to developing mass spectrometric methods applicable to characterization of DNA modifications. It has not been possible here to acknowledge individually the many investigations devoted to structure determination of genotoxin adducts, but these are categorized and described in the review by Chiarelli and Lay (H68). Some more recent work of this kind has been described by Gross and collaborators (H118, H119). Despite all of this effort, it is sobering to acknowledge that a recent review article (H120) on detection of DNA damage (mostly oxidative and photochemical) hardly dealt with mass spectrometry at all, other than the GC/MS methodologies referred to above. With respect to the sequencing of oligonucleotides containing one or more modified bases, mass spectrometry clearly has a unique role to play (H112) although the available sensitivities are still well below those routinely available using classic chemical and biochemical approaches. In the complementary role of detection and quantitation of modified mononucleosides liberated by total hydrolysis of the DNA, mass spectrometry again has a role to play but even here, as pointed out in the excellent critical review by Farmer and Sweetman (H70), sensitivity is still an issue although GC/ MS with NCI does seem adequate in applications to assessment of oxidative damage (higher levels of modification). The potential advantages, derived from the much greater information content provided by analysis using MS or MS/MS, will not be fully realized until sensitivities can be improved by orders of magnitude. In this regard, the demonstration by Greig et al. (H121) that negative ion microelectrospray can provide an increase by more than two orders of magnitude in the integrated ion abundance normalized to the amount of infused sample, relative to conventional electrospray techniques, is promising. However, it is well to recognize the potential of rival analytical techniques, e.g., laser-induced fluorescence detection for capillary electrophoresis appears to have the required sensitivity (H122) though of course the information content (selectivity) is much lower than for mass spectrometry. Proteins are much more readily available than DNA from organisms exposed to genotoxins and contain a variety of nucleophilic sites which are susceptible to attack by electrophilic forms of the genotoxins. In addition, the stability of many proteincarcinogen adducts is comparable to that of the native proteins, so that analysis of the protein adducts is less challenging and can serve as a surrogate indicator of exposure to genotoxins. Two relatively abundant proteins in mammals, hemoglobin and albumin, have been extensively used for this purpose as described by Farmer and Sweetman (H70). Structure determination of
modified proteins follows essentially standard procedures, using LC/MS/MS analyses of tryptic digests for example, although some adducts are unstable and require special treatment. With regard to quantitation, the most common method is that due to To¨rnqvist et al. (H123), which again employs GC/MS with NCI. The stategy exploits the fact that both R- and β-chains of hemoglobin have valine as the N-terminal residue. A modified Edman degradation chemistry using pentafluorophenyl isothiocyanate yields an N-alkylated pentafluorophenylthiohydantoin as the Edman product from an alkyl-modified N-terminal valine. These Edman products are hydrophilic and are readily separated from the remaining protein (H123). Detection limits as low as 1 pmol of adduct per gram of globulin have been obtained. Examples are described by Farmer and Sweetman (H70). In conclusion, the current state of mass spectrometric technology is adequate for quantitative monitoring of exposure of proteins to genotoxic agents, but this is not true for DNA. The Randerath 32P-postlabeling procedure (H51, H52) does not provide structural information on the adduct, and this is usually obtained by chromatographic comparisons with synthetic standards whose structures have been determined by mass spectrometry, NMR, etc. This gap provides a real challenge to the mass spectrometry community. Sequencing of modified oligodeoxynucleotides is often difficult or impossible for the standard sequencing technologies, and this deficiency provides a similar challenge.
ACKNOWLEDGMENT
We acknowledge discussions and information from many colleagues too numerous to cite individually. A.L.B. is once again indebted to the expertise and talents of Candy Stoner for extensive library searching, transcription, and editing of the entire manuscript. Financial support was provided by NIH NCRR, Biomedical Research Technology Program Grant RR 01614 (to A.L.B.), NIH Liver Center Grant AM27643 (to A.L.B.), and the University of Manchester Institute for Science and Technology.
A. L. Burlingame is Professor of Chemistry and Pharmaceutical Chemistry at the University of California, San Francisco. He is Director of the NIH-supported National Bio-organic, Biomedical Mass Spectrometry Facility and of the core mass spectrometry facility of the Liver Center in the School of Medicine at UCSF. He received his B.S. from the University of Rhode Island and his Ph.D. from the Massachusetts Institute of Technology in 1962. His current interests focus on sequence determination of proteins in tumor cell lysates separated on 2-D gels, and detailed structural studies of posttranslationally and xenobiotically modified proteins, cell surface receptors and glycoconjugates of biomedical importance. He is a member of the American Society for Mass Spectrometry, The Protein Society, and The American Association for Biochemistry and Molecular Biology, and an Elected Fellow of the American Association for the Advancement of Science.
Robert K. Boyd is Principal Research Officer and Analytical Chemistry Group Leader at the Institute for Marine Biosciences, National Research Council, Halifax, Nova Scotia, Canada. He received both B.Sc. and Ph.D. degrees in physical chemistry from St. Andrews University. He has been appointed as a Canadian representative to the Chemical Metrology Working Group of NORAMET, the metrology subcommittee working under the auspices of the North American Free Trade Agreement, and has been elected to the Board of the Canadian Association of Environmental Analytical Laboratories. In 1996 he will complete a two-year term on the Board of the American Society for Mass Spectrometry and a threeyear term on the Grant Selection Committee for Physical and Analytical Chemistry of the Natural Sciences and Engineering Research Council of Canada. He is an Editor of Rapid Communications in Mass Spectrometry and is on the Editorial Board of the Journal of Mass Spectrometry.
Simon J. Gaskell is Professor of Mass Spectrometry and Director of the Michael Barber Centre for Mass Spectrometry in the Department of Chemistry of the University of Manchester Institute of Science and Technology (UMIST). He received his B.Sc. (1974) and Ph.D. (1977) degrees from the University of Bristol. He took up his present position at UMIST in 1993. His research activities are in the development of mass spectrometry (particularly tandem MS) and related techniques, and their application to biological research. Specific interests range from the characterization of modified peptides and proteins to the trace quantification of lipid mediators. He is the cofounder (with R. K. Boyd and P. W. Brooks) and current Chairman of the Lake Louise Workshops on Tandem Mass Spectrometry. He is an Associate Editor of the Journal of the American Society for Mass Spectrometry, Rapid Communications in Mass Spectrometry, and Mass Spectrometry Reviews.
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A1960021U
Analytical Chemistry, Vol. 68, No. 12, June 15, 1996
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652R
Analytical Chemistry, Vol. 68, No. 12, June 15, 1996
