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Mass Spectrometry A. L. Burllngame' Department of Pharmaceutical Chemistry, The Mass Spectrometry Facility and the Liver Center, Universiw of California, San Francisco, California 94 143-0446 Robert K. Boyd Institute for Marine Biosciences, National Research Council, Halifax, Nova Scotia, Canada B3H 32 1 Simon J. Gaskell Department of Chemistty, University of Manchester Institute of Science & Technology, Manchester, UK Review Contents Overview Scope Innovative Techniques and Instrumentation Ionization Techniques-MALDI and Electrospray Time-of-Flight Analyzers in Tandem MS Fourier Transform Ion Cyclotron Resonance (FT-1CR) MS and Quadrupole Trap MS Surface-Induced Dissociation Mass Spectrometric Studies of Noncovalent Interactions Collision-Induced Light Emission Mass Spectrometric Detection for On-Line High-Resolution Separations Ionization Sources and Interfaces for MS as a Detector Separation Techniques Which Can Use MS Detection Decompositions of Peptide Ions Peptides and Proteins Amino Acids Synthetic Peptides and Proteins Bioactive Natural Peptides Posttranslational Modifications Disulfide Linkages GIycosylation Phosphorylation Hemoglobin Variants Low-Energy Collision-Induced Dissociation High-Energy Collision-Induced Dissociation Protein Covalent Modification Gel Electrophoresis Noncovalent Associations Metal Ion Associations Carbohydrates and Glycoconjugates Derivatization and Techniques Oligosaccharides Sulfated Oligosaccharides Periodate Oxidation Lipopolysaccharides/Lipooligosaccharides Glycopeptidolipid Surface Antigens GIycosphingolipids Miscellaneous Applications in Biological Research
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A. OVERVIEW This is the era of biological mass spectrometry, the study of macromolecular science in the overall context of human health and disease. This vigorous new discipline, based upon 034R
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new ways of creating ions of polar and labile biopolymeric species with remarkable ease and efficiency, is redefining a major segment of analytical chemistry. The creation of new instrumentation based on widely varied strategies is underway, conceived to take full advantage of these different forms of ionization. These technical advances are providing the tools to elicit the true nature of macromolecular structure, variation, and heterogeneity with unprecedented levels of insight. Impressive strides have already been made, resulting from a highly productive embryonic symbiosis between mass spectrometrists and biological scientists. The unifying theme underlying these successes concerns the inherent ability of mass spectrometry to detect and define the nature of complex mixtures and heterogeneity in a global sense, prior to any need to isolate individual components for rigorous structural elucidation. In addition, further dimensions of analytical power may be brought to bear from the beginning through use of liquid chromatography and/or tandem mass spectrometry. Many times these methods alone provide the answer sought, thus obviating the need for isolation of an individual component. Electrospray and MALDI techniques now provide the means to carry out such analyses on several levels, from the mixtures of degradation products themselves to the intact molecular weight framework encompassing the biopolymeric entity within which the components must fit. The effectiveness of this joint venture is evident in work on some of the most difficult mixture/structure problems. Contributions from a number of laboratories have focused attention on understanding the amyloidogenic process in Alzheimer's disease through structural studies of the amyloid cores isolated from patient tissues (AI-A6). Plasma desorption (AI-A3), matrix-assisted laser desorption (A4, A5) time-of-flight, and LSI high-performance tandem mass spectrometric (AS, A6) techniques have provided structural information precisely defining ragged termini, as well as primary sequence information not available from earlier use of the Edman degradation. The promise of achieving sequence from much higher mass peptides is indicated by high-energy CID analysis of doubly protonated ions in the 4000-5000 mass range (A6, F187). Further examples showcasing the breadth, versatility, and power of these techniques include sequence analyses of spots isolated from tumor cell cultures by 2-D gel electrophoresis (F242, F243); a large number of identifications of posttranslational and xenobiotic modifica0003-2700/94/0366-0634$14.00/0
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A. L. Burlingame is currently Visiting Professor at the Ludwig Institute for Cancer Research, London, with Professor M. D. Waterfield. He is on sabbatical leave from his position as 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 with K. Biemann in determination of the structure of indole alkaloids. He immediately joined the Department of Chemistry and Space Sciences Laboratory of the Universityof California, Berkeley, as Assistant Professor of Chemistry. He assumed his current responsibilities in 1978. From 1964 to 1973, he was a member of several interdisciplinary scientific teams and committees entrusted with the planning and conduct of the lunar science program and the preliminary examination and distribution of lunar samples from the U.S. Apollo and USSR Luna sample return missions. During this time, as director of the mass spectrometry unit on the Berkeley campus, he pioneered the development of real-time, highsensitivity, high-resolution mass spectrometry, field ionization kinetics, and deuterium difference spectroscopy in NMR analysis. During 19701972 he was awarded a J. S. Guggenheim Memorial Fellowship, which was spent on biochemical-biomedical applications of mass spectrometry with J. Sjovall at the Karolinska Institute, Stockholm. 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 active in the development of techniques and instrumentation required to advance such studies of the machinery of cells. 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. 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 BSc. (1974)andPh.D.(1977)degreesfrom the University of Bristol, the latter under the direction of Professor G. Eglinton FRS. After a postdoctoral Fellowship at the University of Glasgow (with Professor C. J. W. Brooks) he spent nine years as head of the Mass Spectrometry Group at the Tenovus Institute for Cancer Research at the University of Wales College of Medicine. During this period he took a one-year leave of absence at the National Institute of Environmental Health Sciences in North Carolina. I n 1987 he moved to Baylor College of Medicine, Houston, as Professor of Experimental Medicine. He took up his present position at UMIST in 1993. His research activities are in the development of mass spectrometric (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 an Associate Editor of the Journal of the American Society for Mass Spectrometryand is on the Editorial Boards of Biological Mass Spectrometry and Rapid Communications in Mass Spectrometry.
tions of proteins (see discussion in section F); investigations of complex mixtures of antigenic peptides, and the recent identificationof a melanoma-specific peptide antigen (FI 70); antigenic lipooligosaccharides from Gram-negative bacteria which mimic glycosylation on host cell surface glycosphingolipids (G72-G77, G 7 8 4 8 5 ) and Mycobacterial lipoglycosylpeptides (G99-GZ02); the characterization of monoclonal antibodies which recognize neural cell adhesion molecules (A7);and fundamental studies of ensembles of differentially isotopically labeled proteins (F2484'251).
Robert K. Boyd is Principal Research Officer and Group Leader of Analytical Chemistry at the Institute for Marine Biosciences, National Research Council, Nova Scotia, Canada. He was educated at St. Andrews University in Scotland, where he obtained his B.Sc. (Hons. Chem istry, 1959), Ph.D. (physical chemistry, 1963),and an enduring distaste for golf. His Ph.D. thesis work under C. Horrex involved the kinetics of the thermolysis of methyl iodide and a determination of the carbon-iodine bond energy. I n 1962 he undertook postdoctoral studies at the National Research Council in Ottawa, where he worked with K. 0. Kutschke on primary processes in the photochemistry of hexafluoroacetone. After two years in the Central ElectricityResearch Laboratories of the UK Electricity Generating Board, he returned to Canada in 1966as a postdoctoral fellow at the University of Toronto, where he worked with G. Burns on nonequilibrium kinetics of dissociation of diatomic molecules behind incident shock waves. I n 1968 he was appointed Assistant Professor at the University of Guelph, where he rose through the ranks to Full Professor in 1979. His introductionto mass spectrometry came during a 197511976sabbatical year with John Beynon at Swansea when, despite having no instrument available, three papers were published including the algebraic approach to generating linked-scan functions for double-focusing instruments. On his return to Guelph he further developed the theory and analytical applications of the linked-scan approach to tandem mass spectrometry and also initiated physical chemistry studies, including a combined experimental and theoretical approach to diatomic dications and a collaboration with the Swansea group on some fundamental aspects of collisional activation. I n 1984 he was invited to spend the summer at the National Institute for EnvironmentalHealth Sciences, in Research Triangle Park, NC, where the first commercial tandem double-focusing instrument was in process of installation, and developed a method for time-to-mass calibration of the linked-scan of the fragment ion analyzer. I n 1986 he moved to the NRC laboratory in Halifax, where a highperformance-sector/quadrupolehybrid instrument had just been installed to assist programs in biochemical structural analysis and quantitative trace analysis of environmental samples. I n 1989 he became Group Leader and Manager of NRC's Marine Analytical Chemistry Standards Program and shortly after took delivery of one of the first production batch of commercial API instruments designed specifically for LC/MS applications. Since then he has spent most of his time and energy trying to keep pace with a very talented group of younger colleagues, working on highly diverse problems including development of CE-MS/ MS techniques for glycopeptide characterization, quantitative analyses of microalgal toxins in shellfish and finfish, identification of the 85% of the organochlorine load in polluted fish, which is not accounted for by the well-known anthropogenic pollutants, quantitative analyses of higher molecular weight polycyclic aromatic compounds in environmental samples, and occasional relapses into physical chemistry.
In nucleic acid research, identification of a non-purine, non-pyrimidine ribonucleoside (Archaeosine) located at position 15 in archaeal tRNA serves to remind us of the longstanding domination of mass spectrometry in this field (A8). In addition,thedetection of RNA transcriptsto 142nucleotides (A9)and the sequencecorrelation of oligonucleotides as large as synthetic AsT20 (AIO) represent impressive technical achievements using both infrared MALDI and electrospray ionization with Fourier transform mass spectrometry, respectively. In glycobiology,these mass spectrometric techniques have already played a major role in bringing analytical clarity and scientific strength, signaling an end to the previous state of structural confusion. It is clear that protein chemists can no longer get away with dumping the glycosylation down the drain and that the carbohydrate chemists must find out exactly what structure is carrying the biological recognition, what it is attached to, and just where it is attached (G8). In addition, subsequent efforts aimed at identification of ligand function are also being addressed. Thus far it is obvious that major new territories are being explored which hold secrets of structure and function previously inaccessible. Analytical Chemistry? Vol. 66, No. 12, June 15, 1994
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The message is now out and many of these notions have begun to capture the imaginations and whet the appetites of the biological scientists themselves (AI I, A12, F.5, FI66).In their hands parts of the field will diverge and begin to take on a certain life of their own. This has already happened to a large extent in biotechnology, especially concerning the need for quality control of recombinant therapeutic agents. Early forays seek naturally to adopt aspects of mass spectrometry which complement the existing Edman sequencing and PCR cloning, using simple, robust instrumentation (AI2, FI66). For example, in conjunction with a PCR-based strategy, 20 of some 40 subunitsof NADH:ubiquinoneoxidoreductasehave been cloned, with verification of the expressed protein being established by electrospray measurements of their molecular weights. N-Terminal acetylation and myristylation was present on nine of them, and one is modified in a way as yet unknown. Meanwhile, the biological mass spectrometristsunderstand that increasingly advanced generations of ion optical systems are in development which will overcome many limitations of the earliest, inexpensive instruments. These new instruments will facilitate work on the more challenging and complex problems by providing better sensitivity and accuracy with extended mass range and Ksolution. On a different tack, accelerator mass spectrometry has been developing rapidly as well and is now providing unique sensitivities (orders of magnitude greater than scintillation counting) for the detection and measurement of long-lived important radionucleotides, including 14Cand 3H(AI 3). This opens yet another dimension of opportunities, even including using tracers in humans. The issueswhich have been raised by reports of noncovalent interactions are qualitatively in their own league for the present (F20). They are provocative and, in the long run, will probably reveal basic information of fundamental importance regarding the nature of ligand/macromolecular recognition. To avoid being overly enthusiastic about our own progress in macromolecular science, it might be worth noting that plasmid DNA containing retinoic acid/thyroid hormone chimeric receptors and appropriate reporter constructs may be transvected directly into living animal wound epidermis tissue in situ by fast DNA coated gold microparticle implantation, and their subsequent expression monitored by immunofluorescence (AI 4 ) . And to keep things in perspective, bear in mind that, "The mass spectrometer has made unique contributions to post-war nuclear physics, ... proved a useful tool in oil analysis and is showing promise in the chemical analysis of solids ... and has made the occasional errands of mercy into several other areas of scientific endeavorn (A15). B. SCOPE As always, some degree of selectivity in these reviews is essential, and inevitably our selection of topics, and references cited within each topic, cannot possibly encompass the entire literature. A serious effort has been made to treat the subject material which seemed to us to represent the most interesting applications and newer methods, to the limited degree of detail which space permits. Accordingly, the reader must consult the cited literature, and other references cited therein, for a full appreciation of the scientific content. 63BR
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Tandem mass spectrometry has been an important feature of these reviews for some time, and indeed a major treatment of this subject was included in the 1992 edition ( B I ) . Accordingly, we felt that it was not an essential topic to be treated in detail in 1994. Instead, the roleof mass spectrometry as a detector for chromatography and other separation techniques, such as capillary electrophoresis, was chosen as a topic of considerable current importance which has not been covered recently in these reviews in a systematic fashion. This material appears as section D and necessarily is slanted more toward quantitative analysis. The traditional commentary on innovative techniques and instrumentation (section C) has always represented the idiosyncratic views of the writers of this review, and the present version is no exception. These reviews appear in a distinguished journal for analyticalchemistry, so section C has been written from the viewpoint of the analytical chemist or biochemist who needs techniques which will provide information on chemical or biochemical problems. That is to say, we do not regard mass spectrometry as a scientific end in itself, but as an important analytical tool. Elegance and imagination, in this utilitarian context, are of value only to the extent that they have led to useful analytical techniques which provide data on demand for often scarce and precious samples. Accordingly, we have, in section C, attempted to give a hardnosed but fair assessment of the degree of analytical utility of some important techniques at the present stage of development. Of course, these opinions may well be controversial (and no apology is offered for this) and, in any case, do not preclude the possibility that some or all of the perceived shortcomings of technique X will be overcome before the next edition of this review is written. Disputes about nomenclature can often degenerate into dry-as-dust pedantry. However, we make no apology for the following comments on the use of the term "accurate mass" in mass spectrometry. Traditionally, this terminology has been used to refer to measurements of ionic mass with uncertainties in the rangeof 1-10 parts per million (ppm). In most cases, the uncertainties in such measurements are dominated by random errors, since sources of systematic error in such measurements are generally well understood and can be accounted for. Therefore, the term "uccurate mass measurementn is something of a misnomer, since the figure of merit which characterizes such measurements is actually the precision. The accuracy is a measure of the deviation of the best experimental estimate (usually the mean) of the quantity (mass in this context) from an assumed "true" value. The interplay between these two concepts in the context of mass measurement is well explained in an editorial written by Gross (BZ), quoting work of the ASMS Committee on Measurements and Standards on the use and misuse of such measurements by the organic chemistry community in confirmation of atomic composition. This editorial should be required reading for the current biooriented mass spectrometry community also. It has become commonplace to use the term "accurate mass measurement" to refer to measurements with precision as low as 1 in IO3, e.g., in MALDI-TOF spectra discussed in section C, in which the peaks are characteristically broad and often asymmetric. Quite apart from ambiguities in how such a peak should be mass-measured (e.g., where to
start and stop the centroiding process, at 5%, lo%, or what, of peak height), there is the possibility that such a broad peak could be an unresolved composite. Efforts toattain high mass resolution are directed at exactly this point, to maximize the reliability of the mass measurement. We believe that the concept described by the commonly used term "accuratemass measurement- would be better described as "exact" (B2), even though the most appropriate term would use "precise" as the descriptor. Equally important, we would urge the mass spectrometry community to take pride in the achievements represented by the availability of instruments permitting routine measurements of ion mass with uncertainties in the low ppm range and to restrict the use of such terminology to performance of this quality. The achievementsof, for instance, the MALDI-TOF and electrospray techniques discussed in section C are sufficiently impressive that they do not require embellishment with unjustifiable claims to the same levels of mass measurement performance as are achieved using techniques which cannot begin to approach their mass range. As has been the case in the last few reviews in this series, the overwhelming emphasis in those sections dealing with applications of mass spectrometry is on problems in analytical biochemistry for a wide range of biopolymeric compound classes, toxicology, clinical chemistry, etc. This emphasis reflects the direction which the disciplinehas taken increasingly since 1981, the date of invention of fast atom bombardment (FAB) ionization (B3). Prior to that date, environmental problems were probably the single most important application area for mass spectrometry, particularly for GC/MS techniques. The more recent development of ionization methods which intrinsically apply to flowing liquid solutions, as discussed in section D, raises the possibility that previously intractable problems of environmental chemical analysis might now be amenable to LC/MS procedures. It will require some time, and some well-documented successes, to persuade regulatory agencies that quantitative analyses by these new LC/MS techniques are sufficiently reliable for acceptance in official protocols. Mass Spectrometry Reviews (B4) continues to provide timely reviews of topics both fundamental and applied; the "review of reviews" section contributed by Budzikiewicz is particularly valuable. Reports of the 40th and 41st ASMS Conference on Mass Spectrometry and Allied Topics have been published (B5, B6), and the 42nd through 45th conferences (1994-1997) will be held in Chicago, IL, Atlanta, GA, Anaheim, CA, and Kansas City, MO. The 13th International Mass Spectrometry Conference will be held in Budapest, Hungary, August 29-September 2, 1994. The Third International Symposium on Mass Spectrometry in the Health and Life Sciences will be held in San Francisco September 13-18, 1994 (Fa). The Montreux Conference on LC/MS will be held in Montreux. Switzerland, November 9-12,1994. Volumes of interest, which appeared in the two years since the previous edition of this review ( B I ) , include Mass Spectrometry for the Characterization of Microorganisms, edited by Fenselau (B7); Mass Spectrometry of Lipids, a monograph written by Murphy (B8); Mass Spectrometry: Clinical and Biomedical Applications, volume 2, edited by Desiderio (B9); Time of Flight Mass Spectrometry, edited by Cotter (BI 0); Liquid Chromatography-Mass Spectrom-
etry, by Niessen and van der Greef ( B I I ) ; Methods and Mechanisms for Producing Ionsfrom Large Molecules, edited by Standing and Ens (812);and Biological MassSpectrometry Present and Future, edited by Matsuo, Caprioli, Gross, and Seyama (BI 3). The latter volume is a collection of 36 articles by experts who presented invited lectures at the Kyoto '92 International Conference on Biological Mass Spectrometry, held September 20-24, 1992, in Kyoto, Japan. It includes chapters on ionization (five chapters), mass analysis (six chapters), structure methods (four chapters), peptides and proteins (seven chapters), oligosaccharides and lipids (five chapters), nucleic acids (one chapter), xenobiotics (two chapters), environmental and endogenous toxic compounds (one chapter), and ana.lytica1 and organic chemistry (four chapters). A volume of abstracts of the contributed papers presented at Kyoto '92 has also been published (B14). Two special volumes of the International Journal of Mass Spectrometry and Ion Processes (BI 5 ) contain the invited lectures presented at the 12th International Conference on Mass Spectrometry held in August 1991 in Amsterdam. A more recent special issue of the same journal @Id), edited by Schlag, is dedicated to timely reviews of time-of-flight mass spectrometry and its applications. A large number of specialistchapters on mass spectrometric techniques continue to be contributed tovolumes dealing with biochemistry and other areas of interest. We apologize in advance to those authors whose reviews are not mentioned below; it is possible to include only a partial listing here. These include articles by Hunt et al. (B17) on mass spectrometric methods in protein sequencing; by Pleasance and Thibault on coupling capillary electrophoresis to mass spectrometry ( B l 8 ) ; and by Settineri and Burlingame on mass spectrometry of carbohydrates and glycoconjugates (BI 9 ) . Other reviews of interest, other than those published in Mass Spectrometry Reviews (B4), appear in refereed journals devoted to mass spectrometry and other aspects of analytical chemistry. Some examplesnot included in the specialvolumes already mentioned include an illuminating account of the development of matrixassisted laser desorption and ionization (MALDI) by Beavis (B20);a comparison by Wang and Chait (B2I) of the relative merits of MALDI and electrospray mass spectrometry in analytical biochemistry; scholarly reviews of mechanistic aspects of FAB and of electrospray ionization by Sunner (B22) and Kebarle and Tang (B23),respectively; an overview of the utility of atmospheric pressure ionization techniques (including electrospray and its variants) in LCMS by Allen and Shushan (B24);a review of time-of-flight analyzers by Cotter (B25); reviews of Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry by Marshall and Grosshans (B26) and by Buchanan and Hettich (B27) and of quadrupole ion traps by Cox et al. (B28) and by Schwartz and Jardine (B29); reviews of the coupling of capillary electrophoresis with mass spectrometry by Smith et al. (B30) and by Niessen et al. (B31), and of coupling thin-layer chromatography to mass spectrometry by Busch (B32)and by Martin et al. (833). Finally, an interesting review of the structures of Archaebacterial membrane lipids discusses their natural resistance to oxidation and esterases, which may be of advantage in the context of applications in biotechnology (B34). Analytical Chemistty, Vol. 66, No. 12, June 15, 1994
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C. INNOVATIVE TECHNIQUES AND INSTRUMENTATION Mass spectrometry is one of the most important experimental tools currently available to the chemistry and biochemistry communities. It cannot be said to be a scientific end in itself. The lasting scientific value of efforts in development of novel instrumentation and techniques must ultimately be decided in terms not of the ingenuity, imagination, and persistence which have been devoted to them, but of the useful information they provide. Mass spectrometry provides information to the analytical chemist concerning either molecular composition and structure of an unknown compound or the concentration of a target analyte present in complex matrices. Mass spectrometry also continues to provide a valuable experimental approach for physical and physical organic chemists interested in the energeticsof gaseous ions and their interactions with neutral molecules, surfaces, electromagnetic radiation, etc. Instrumentation developments which succeed in providing new or better information are to be encouraged, but when superseded by better methods or when it becomes apparent that they have run their course, the ingenuity devoted to refining them would be better applied elsewhere. Like all provocative statements, the preceding paragraph contains some truth (not to mention truisms) and some oversimplifications. An example of instrumentation development, treated at some length in section D below, is that of LC/MS coupling. As discussed in a recent review by van der Greef and Niessen ( C I ) , some 25 LC/MS interfaces have been described over the past 20 years or so, but only 5 are actively used today and even fewer are still the subject of much development effort. This is an example of an area in which short-term practical concerns of the professional analyst, about the actual performance of a particular device relative to that of its rivals, have dictated the amount of research effort devoted to its further development. Of course, this criterion can be taken too far. Who would have predicted 10 years ago that the phenomenon of electrospraying of liquids (assuming that any significant number of mass spectrometrists had ever heard of it) would have had such a profound effect on LC/MS experimental practice? Nonetheless, in this section we have assumed the role of agents provocateurs in the service of the viewpoint expressed in the opening paragraph. We have been led to adopt this attitude in the present section by our own bewilderment (which we suspect is shared by many others) over the competing and often contradictory claims of rival techniques for ionization, mass analysis, ion activation, detection, data analysis, etc. This biennial review is directed primarily at analytical chemistry. Therefore, a negative or ambivalent answer to the hard question as to whether or not a specific technique is likely to provide useful analytical information, or whether this information would be more easily or better obtained by some other method, does not preclude the possibility that the technique might be a valuable tool in physical chemistry, for example. IonizationTechniques-MALDI and Electrospray. These two techniques have dominated developments in mass spectrometry over the last few years, certainly with respect to biological applications. The implications for on-line MS 830R
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detection for separatory techniques are discussed in section D. Inevitably electrospray and its variants are paid considerably more attention in that particular context, but here the two techniques are discussed on a more equal footing, that of analysis of prepurified biochemical and other samples. Matrix-assisted laser desorption/ionization is a development of the older LDI technique, in which illumination of a solid sample with a short intense light pulse can produce molecular ions, or protonated or cationized molecules, from thermally labile molecules. Sensitivitiesin the picomole range have been reported (CZ-C4). The efficiency of LDI is a complex function of many variables, including the wavelength and power density of the laser radiation, the incident angle of the radiation, the optical absorption characteristics of the sample, the thickness of the sample layer, and the nature of the substrate surface (CS-C8). Although exceptions exist (C4, C8), LDI appears to be most efficient (C9) when the laser wavelength matches an intense absorption band of the sample, but direct absorption of radiation by the analyte can lead to fragmentation and failure to obtain molecular mass information. The idea of dispersing the sample in a radiation-absorbing matrix, in order to limit radiation damage to the analyte, appears to have been introduced first by Tanaka et al. (CIO), who used a liquid matrix in which was suspended a fine metal powder of 30-nm particle size, and by Karas and Hillenkamp (CII), who used a solid matrix to obtain spectra of mellitin and other peptides of molecular mass up to 3000 Da. The work of Tanaka et al. (CZO) produced LDI/TOF spectra of proteins up to 35 kDa. The history of how this remarkable early achievement provided a key impetus to further development of MALDI, as currently practiced, has been described by Beavis (CI2) in an excellent article. Tanaka’s work (CZO) is discussed further below, in the context of a possible mechanism for the role of the metal particles. The use of solid matrices for MALDI, the current standard practice introduced by Karas and Hillenkamp (CII) and later by Beavis and Chait (CI3),was quickly shown to be capable of providing spectra of proteins of even higher molecular masses than those demonstrated by Tanaka et al. (CIO).Informative reviews of the practice and achievements of solid-matrix MALDI have been published by these workers (CI4-CI 7). This innovation was somewhat analogous to the introduction of a liquid matrix to the practice of secondary ion mass spectrometry (FAB-MS). The mechanisms involved in MALDI are still obscure, and the method has been developed along empirical lines (again somewhat analogous to the case of FAB/LSIMS). A particularly detailed discussion of possible models and mechanisms for MALDI has been contributed by Vertes to a volume edited by Standing and Ens (CI8), dedicated to more fundamental studies of methods for producing gaseous ions from large thermally fragile molecules, and which includes several highly informative contributions on MALDI and related subjects. A brief summary of current experimental and theoretical work on the mechanisms proposed to underlie MALDI has been included by Heise and Yeung (CI9) in their recent paper describinguse of a laser-inducedfluorescence technique to study the effect of sample morphology on the plume dynamics.
Matrix selection for MALDI is one of the essential features which is still poorly understood. Matrices of highly similar structures and optical absorption characteristics can yield large differences in MALDI efficiencies for the same compound, while different compound types (e.g., DNA and proteins) behave very differently in the same matrix under the same conditions. The matrix selection problem was addressed by Beavis (C12)in an extensive study of more than 300 potential matrices, of which only 7 proved acceptable. Sample preparation also appears to have a large effect on the outcome. As usually prepared by the “dried droplet” method, whereby a droplet of solution of mixed matrix plus analyte is deposited on a metal substrate and allowed to dry, samples often contain a higher analyte concentration around the edge (C.20).For this reason the MS intensity can vary widely with position of laser irradiation of the dried droplet (C20, C21). It is possible to use plastic rather than metal substrates ( ( 2 2 ) to enable realization of MALDI in transmission geometry, rather than in the more usual reflection geometry where laser illumination and mass analyzer are on the same side of the sample. Direct MALDI of proteins electroblotted on to polymer membranes, following SDS-PAGE separation, has been demonstrated recently (C23)to give superior results using an infrared rather than a UV laser. Use of etched silver foil substrates has shown better uniformity of ion production with respect to irradiation position on the dried droplet (C24). It has been claimed that MALDI of proteins is tolerant of (C25) or immune to (C26) high concentrations of salts, buffers, and other involatile components (e.g., dimethyl sulfoxide, glycerol, urea, sodium dodecyl sulfate) commonly present in protein preparations in order to increase stability and/or solubility. However, in the experience of the present authors this degree of tolerance is highly variable, and indeed Xiang and Beavis (C27) recently published some excellent work devoted to devising methods to permit removal of such contaminants from protein samples prepared for MALDI analysis. This very recent work (C27) used, in an exemplary fashion, existing knowledge of more fundamental aspects of cocrystallization of analyte and matrix on a stainless steel substrate (probe tip) to guide the development of a sample preparation technique which proved far superior to the “dried droplet” method. The resulting polycrystalline films adhered firmly to the steel surface and could be washed vigorously to remove contaminants, leading to considerable improvement in performance. Previous efforts at improved sample preparation included the approach of Speir and Amster (C28), who used a UV-absorbing matrix in a sandwich arrangement between the metal substrate and the outer analyte layer. Hutchens and Yip (C29) used an interesting and provocative approach by modifying the substrate surface to provide a chemically bonded matrix onto which the analyte could be adsorbed. Unfortunately it has not been possible (C27)to reproduce the original results (C29), for reasons which remain unclear. A systematic investigation of the effects of different drying techniques on the efficacy of the “dried droplet” method of sample preparation (C30) indicated that vacuum drying is faster and provides better ion yields and resolution in MALDI/TOF spectra, though no investigation of contaminant tolerance was reported. Chan et al. (C31, C32) investigated the problem of the very intense ion signals derived from the matrix, observed below m / z 500
or so. Beavis and Chait (C13) had employed pulsed deflector plates to suppress theselow-mass ions which Chan et al. (C31, C32) showed were also suppressed over a narrow range of matrix/analyte ratios. Unfortunately, this optimum range was observed (C32)to be compound dependent, with a marked dependence on analyte molecular mass. Very recently Quist et al. ((233) directly measured total laser ablation yields of neutral species from a MALDI matrix, using a quartz microbalance technique. The neutral species yields were compared with those of positive ions as a function of laser fluence, angle of incidence, etc. Two fluence thresholds for total positive ion formation were observed (C33),the lower of which was interpreted in terms of surface desorption while the higher threshold was believed to indicate onset of desorption from a volume. Only the latter threshold gave rise to significant analyte ion signals. A rather different approach to the problems of sample preparation for MALDI, and of intense matrix ions and/or of analyte-matrix adducts, has been described by Wahl et al. (C34),who did away with the solvent matrix altogether and deposited the analyte onto a 10-nmthickgold filmon a glass plate. The film thickness was tailored so that its absorbance maximum matched the fundamental wavelength (1064 nm) of a Nd:YAG laser. The mechanism is believed (C34) to involve efficient thermal desorption of the analytevia local resistive heating of the gold film by induction under the coherent rapidly varying electric field of the radiation. In this regard it is interesting to note, as pointed out by Wahl et al. (C34), that the very early MALDI experiments of Tanaka et al. (CIO)used a liquid matrix with a finely dispersed metal powder of particle size (about 30 nm) much smaller than the wavelength of the laser radiation (337 nm). Tanaka et al. (CIO)proposed a similar mechanism for their procedure. Rather similar experiments (C35),using a 27-nm aluminum film with second harmonic output (532 nm) of a Nd:YAG laser, were interpreted in terms of a particular mechanism, viz. surface plasmon excitation (collective electron excitation (C36)) for transfer of the laser power to thermal modes of the metal substrate. The thin metal film approach (C34, C35) certainly avoids the problems of formation of matrix adducts, but thus far has been demonstrated only for fairly small polar molecules (Rhodamine B, gramicidin S , guanosine, cellobiose) with no investigation of the tolerance for common contaminants, as discussed above. On a somewhat different note, a recent paper (C37) has described initial experiments devoted to discovery of appropriate matrix materials and other experimental conditions for the use of a tunable (free-electron) infrared laser in DNA sequencing, an area in which more conventional MALDI techniques have thus far proved inadequate relative to accepted biochemical methods. The preceding discussion has tried to summarize the development of MALDI, without attempting to provide an encyclopedic listing of all papers published on the subject. It is appropriate to comment briefly on the mass analysis of ions produced by MALDI. By far the most common analyzer used for this purpose has been the time-of-flight (TOF) mass spectrometer, as described in a timely review due to Cotter (C38), and most of the ensuing discussion will focus on TOF analysis, A more detailed discussion of the use of TOF analyzers in tandem mass spectrometry is included later in Ana&tical Chemistry, Vd.66,No. 12, June 15, 1994
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this section. The characteristically broad and often asymmetric peaks (mass resolution typically 200-500, based on full width at half-maximum (fwhm), decreasing to about 50 for molecular masses above 100 kDa or so), observed in MALDI-TOF spectra of proteins and other large molecules, are believed to reflect in part the intrinsic axial velocities of the ejected ions which appear to be largely independent of the molecular masses (C39-C41). In addition, unimolecular decay along the TOF flight tube is believed to play an important role in determining peak widths (C42). Fragmentations of large organic ions such as proteins appear to be qualitatively different from those of smaller peptides, as emphasized in an intriguing paper by Demirev et al. (C43). Whereas small peptides yield structurally informative fragments arising from backbone cleavages, larger protein ions fragment mainly via nonspecific losses of small neutral groups to give a continuous background of low-intensity uninterpretable fragments. It is interesting to note that Demirev et al. (C43) also conducted time-delay experiments and found that the desired backbone cleavages became more competitive at longer times after ionization. These conclusions (C43) were drawn from experience based upon FAB and plasma desorption ionization, both known as “hard”ionization techniques. However, Spengler et al. (C42) report similar conclusions concerning dominance of small neutral group losses for protein ions formed by MALDI, which is thus clearly not a “soft” ionization technique in the sense that electrospray and field ionization can be so designated. These observations (C42, C43) are in accord with early general predictions of Bunker and Wang (C44), who considered a theoretical model for fragmentation of long-chain polymers taking into account variations in the frequency factors in the dissociation rate constants for bonds in different positions along the chain. This model (C44)predicts preferential losses of small groups from the ends of the molecule as the chain length increases, compared to midchain cleavages which dominate for shorter chains. The “nonsoft” natureof MALDI thus contributes to the limited resolution obtainable in MALDI-TOF, but is also a driving force in current developments of methods for tandem mass spectrometry using TOF analyzers. These developments are discussed in more detail below, but it is appropriate to mention here that those precursor ions whose fragmentations have been investigated in TOF instruments are fairly small and/or contain relatively weak bonds, consistent with the considerations of Demirev et al. (C43). An interesting example is provided by the oligosaccharide portions of glycopeptides, investigated by Martin et al. (C45). Modern reflectron designs can significantly improve MALDI-TOF performance (C38,C42)for what are by current standards low molecular mass compounds, but are of little use above about 10 kDa. This limited resolution obviously places corresponding limits upon the abundance sensitivity of the technique with respect to detection of protein ragged ends or of glycoform heterogeneity, etc. (The concept of “abundance sensitivity” is a very old one in mass spectrometry, originating in work on detection of rare isotopes close to abundant isotopes in the mass spectrum (C46).) The same problem of axial kinetic energy of ions limits the performance of other mass analyzers in combination with MALDI. Thus a doublefocusing sector instrument equipped with an array detector 84QR
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was able to achieve a resolution of 1000 (fwhm basis) for bovine ubiquitin (molecular mass 8565 Da) at picomole sensitivities (C47). Similarly, marriage of MALDI with FT-ICR requires trapping of the fast ions, recognized as one of the problems to be overcome (C48, C49). Castro and Wilkins (C50) have reported MALDI-FT-ICR spectra of bovine insulin (5734 Da) with a resolution (fwhm) of 30 000. These workers interpreted their observations on the resolutionenhancing effect of the matrix in terms of cooling the analyte ions sufficiently that stabilization via infrared emission could compete with unimolecular decay, thus providing the requisite population of long-lived ions necessary for high-resolution measurements in the ICR cell (C50). Buchanan and Hettich (C51)have reviewed these problems in MALDI-FT-ICR and emphasized that the highly nonthermal axial energies of the ions present a special challenge. Similar ion trapping problems were encountered by Chambers et al. (C52) in coupling MALDI with a quadrupole ion trap, and these may have accounted in part for an unexpected large drop in both sensitivity and resolution above about m / z 3000. There are many examples in the literature of MALDITOF techniques providing key information on real-world unknown samples, and these are covered in later sections of this review. Just one will be mentioned briefly here, since it illustrates a more general point. Horiuchi et al. (C53)isolated CAMP-regulated phosphoproteins from bovine caudate nuclei and characterized them using standard biochemical techniques. One of these proteins was designated ARPP-16, the suffix denoting the molecular mass (16 kDa) determined using a gel electrophoresis method (SDS-PAGE, usually considered (C54)to provide molecular masses accurate to within 5-10%). However, the protein sequence, deduced (C53)from that of the cDNA since the N-terminus of the protein was blocked by an unknown moiety, indicated a molecular mass of 10 665 Da and thus a discrepancy of some 50% from the SDS-PAGE value. Later (C14) a MALDI-TOF spectrum of the ARPP16 protein indicated a molecular mass of 10 709 Da, only some 44 Da larger than that predicted (C53)from the cDNA sequence and consistent with the presence of acetyl as the N-terminus blocking group. Although the MALDI-TOFmass spectrum was not available to the original workers in this case (C53),it does provide a dramatic example of the value of the technique which provided the information in a few minutes while consuming only 1 pmol of sample (C14). This notable success, however, is no reason to lose perspective on the limitations of this (or any other) technique. In the cited example, the MALDI-TOF method succeeded in establishing the molecular mass where SDS-PAGE failed badly, but the level of confidence in establishing protein purity was restricted by the abundance sensitivity limitations discussed above. For example, recombinant proteins intended for human pharmaceutical use must meet stringent regulatory criteria of purity and homogeneity which would not be approached by a MALDI-TOF spectrum. This is not to say that MALDITOF would not provide an invaluable tool during development of such a product, but simply emphasizes that all techniques have both limitations and strengths which must be evaluated in each particular context. Thus, it is true that MALDI-TOF has been shown both to provide molecular mass information for proteins at the picomole level and to be capable of providing
mass accuracy of 1 in lo4with use of a more lengthy calibration procedure incorporating internal standards. However, at least in our experience, it is not possible to routinely achieve these specifications simultaneously. In order to achieve 0.01% mass accuracy it is necessary to work near the laser fluence threshold for ion production, where ion yields are much lower (C23) and where it is correspondingly more difficult to achieve high sensitivity. However, it is fair to say that it appears to be possible, for peptides up to 2000 Da or so available at the level of a few picomoles, to obtain molecular masses accurate to within 2 Da with relative ease and speed. This alone is an invaluable biochemical tool. Electrospray ionization and its variants are also widely used in characterization of proteins and other biological molecules and indeed are now almost routine in this field only some six years after the initial reports of Fenn et al. (C.53, though CF-FAB will continue to be widely used on instruments without API capability. In this regard one should note the remarkable data reported by Kenny and Orlando (C56),who used CF-FAB and a tandem double-focusing (four-sector) instrument equipped with an array detector to obtain MS/MS data on peptides in the molecular mass range 9002000 Da, at sample consumption levels in the range 1-6 pmol. Later sections of this review will describe successful applications of electrospray mass spectrometry. In the present section, more general practical concerns are addressed. One great advantage of electrospray (shared to a lesser degree by CFFAB) is its ready compatibility with separatory techniques such as LC and CE (see section D below, where key references are given) due to its intrinsic nature as a technique for flowing liquid solutions. This strength can also be regarded as a weakness, relative to MALDI-TOF, in the context of methods for providing quick occasional checks on molecular mass, e.g., for products from a peptide solid-phase synthesizer, since the solvent flow to the electrospray source must be established and stabilized prior to sample injection. However, the quality of the information obtainable using electrospray ionization far exceeds that from MALDI in its current state of development. Even when used in conjunction with a single quadrupole mass filter without on-line chromatographic preseparation, electrospray can in principle provide much better resolution and abundance sensitivity than MALDITOF. When coupled to sector instruments, these figures of merit are improved even further (C57), and lower chargestate species can be detected and studied. If array detectors are used, the instrumental sensitivity is also dramatically improved over that obtainable using scanning instruments ((2.58). Similarly, the efforts of Smith et al. (C.59-C6l), to develop high-sensitivity, low-flow rate electrospray techniques for FT-ICR and CE/FT-ICR (see section D) are extremely promising if they can mature into a routine laboratory tool. Of course, the cost of both high-performance sector and FT-ICR instrumentation is much greater than that of a MALDI-TOF mass spectrometer, and the latter also has the advantage of a theoretically unlimited mass range (though limited in practice by detector sensitivity). In addition electrospray ionization is highly susceptible, appreciably more so than MALDI, to suppression effects by reagents commonly employed in protein chemistry and other branches of biochemistry. When used in LC/MS or CE/MS (section D)
such electrospray suppression effects are of little consequence. However, the present discussion is restricted to analyses of prepurified samples as in current MALDI-TOF practice, and careful cleanup procedures are necessary. A systematic investigation of the effects of common contaminants on electrospray efficiency for peptides, and a convenient on-line cleanup method, have been described by Kay and Mallet (C62). Electrospray ionization has mostly been used for hydrophilic proteins and other biopolymers, while hydrophobic species such as membrane proteins have seldom been analyzed in this way. This is again due mainly to incompatibility between electrospray ionization and the detergents and/or salts required to retain such proteins in solution in aqueous methanol or acetonitrile. Recently, Schindler et al. (C63) developed sample-handling protocols and solvent systems that are compatible with electrospray mass spectrometry and also retain highly hydrophobic proteins and peptides in solution. The electrospray ionization process itself is extremely “soft”, thus optimizing the probability of obtaining molecular mass information. However, an advantage of electrospray ionization is that the degree of fragmentation observed in the mass spectrum is under the direct control of the experimentalist. This feature arises from the fact that, during the transfer of the ions from atmospheric pressure to the mass spectrometer vacuum, the ions must traverse a region where the background pressure is sufficiently low, and the mean free path sufficiently high, that the translational energy corresponds closely to the potential energy drop generated by the applied electric field. At much higher pressures the collision rate is such that the ions never approach this limit, but rather move with their electrophoretic drift velocities. However, the pressure in the interface region is sufficiegtly high that activating collisions occur. This feature was traditionally exploited in atmospheric pressure ionization sources to remove water and other solvent molecules clustered to the ions emerging from the source, but can also be used to control the degree of ion fragmentation. For example, Starrett and DiDonato (C64) were able to achieve 5 ppm mass accuracy on fragment ions produced in this manner from peptides, using a double-focusing instrument. The additional analytical selectivityaccessiblethrough exploitation of this feature is exemplified by the work of Huddleston et al. (C65) and of Ding et al. (C66) on selective detection and characterization of phosphorylated peptides in negative ion electrospray ionization LC/MS, via diagnostic ions at m / z 63 and 79 corresponding to POz- and PO3-, respectively. A similar LC/MS strategy for proteolytic digests of glycoproteins, exploiting the tunable degree of fragmentation available in electrospray sources, has been used to obtain glycoprotein mass maps which indicate both peptides and Nand O-linkedglycopeptides (C67-C71). This ability todirectly control the degree of fragmentation appears to be a major advantage of electrospray over MALDI and FAB which is not often recognized. The most important general conclusion from the foregoing discussion of MALDI and electrospray is that, as in most questions, the situation is complicated and there are no universal easy answers. In the realm of biological mass spectrometry each of MALDI, electrospray, and FAB has its own niche. There is little point in extolling the virtues of one technique in isolation from the overall perspective of the present Analytical Chemistty, Vol. 66,No. 12, June 15, 1994
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section, viz. that mass spectrometry is an invaluable scientific tool for chemical and biochemical analysis, not a scientific end in itself. While it is clear, as pointed out by Wang and Chait (C26), that MALDI-TOF and electrospray are complementary techniques for biochemical analysis, and it is highly desirable to have both available if possible, not all laboratories can afford to acquire both simultaneously and one is then faced with the question of priorities. There is a preponderance of evidence in the current literature to the effect that electrospray ionization, with even a simple quadrupole analyzer, can contribute significantly to more problems than can MALDI-TOF, at least in the current stages of development. However, the relative ease of operation of the latter is also a significant consideration. It is also appropriate to add a caveat on behalf of static SIMS, a technique of high intrinsic sensitivity whose potential has been reemphasized by Mantus et al. (C72). Time-of-Flight Analyzers in Tandem MS. The essential feature of tandem mass spectrometry is the establishment of connectivity between precursor and product ions. In this section we review the use of time-of-flight analyzers, either alone or in combination with other types of mass spectrometer, for the purpose of examining ion fragmentations via the determination of precursor/product relationships. There are three reasons for the recent attention to the utility of TOF analyzers in tandem MS: (i) TOF instruments are (in comparison with sector-based instruments) relatively inexpensive; (ii) TOF analyzers are well matched to pulsed ionization techniques such as laser desorption; and (iii) the TOF analyzer enables the rapid repetitive recording of full mass spectra from single pulse ionization events or ion injections. The mass range of the analyzer (which is, in principle, unlimited for TOF) is not currently a relevant concern (though it may become so) since the applicability of tandem MS is limited by the difficulty of inducing (and understanding) the fragmentations of large ions. Point iii merits further discussion. The selectivity of the tandem M S experiment introduces an analytical situation, relatively uncommon in “conventional” MS, in which the detection limit is usually determined by the absolute signal intensity relative to the electrical noise, rather than by the ratio of signal to chemical background. (Exceptions do exist, e.g., the demonstration by Falick et al. (C73) of the effect of the “peak-at-every-mass”background, in FAB mass spectra, on fragment ion spectra of precursor ions present at low intensity.) Thus, the limitations of scanning mass spectrometers become apparent, and attention has been drawn to devices benefiting from the Fellgett advantage, Le., improvement in signal/noise ratio derived from simultaneous detection of all or part of a spectrum of ions. The incorporation of array detectors in four-sector tandem mass spectrometers has been reviewed elsewhere ( C 7 4 4 7 6 ) . The extent of the benefit derived from their use is dependent on the range of product ions for which simultaneous detection is achieved ( C 7 4 4 7 8 ) or upon the incorporation of a scanning facility in conjunction with array detection (C75, C79). It is perhaps obvious, but nevertheless worth emphasizing, that in analytical modes resulting in the generation of product ion spectra the continuous selection of the precursor ion species can be achieved using a variety of mass analyzers, and the benefit 642R
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of the Fellgett advantage is solely dependent on the choice of analyzer for the product ions (C80). An alternative approach to the use of array detectors in all-sector instruments is provided by the incorporation of a TOF spectrometer as product ion analyzer (MS2). Used as a conventional mass spectrometer, the TOF analyzer enables the repetitive recording of full mass spectra from single pulse ionization events or ion injections. Equivalently, pulsed introduction of ions into the TOF analyzer as MS2 of a tandem instrument enables the detection of all product ions. Russell and co-workers (C81) described a hybrid instrument of EB/ TOF geometry in which precursor ions, typically of 8-keV kinetic energy, were decelerated into a collision cell floated at 7 kV. Subsequent reacceleration into the grounded TOF region thus resulted in a population of transmitted ions (products and surviving precursors) with kinetic energies in the range 7-8 keV. Initial studies involved the use of pulsed ionization (from laser desorption of analyte). Collisionally activated dissociation (CAD) of protonated angiotensin [M + HI ions yielded a series of products proposed to correspond to consecutive cleavages of the peptide backbone and losses of amino acid side chains. The authors suggested (C81) that the relative prominence of these products might reflect a larger acceptance angle for scattered ions by comparison with that on tandem instruments incorporating a sector instrument as MS2. Additional data are required, however, to substantiate this hypothesis. Some of the issues involved in sector/TOF coupling have been succinctly addressed in a recent note by Clayton and Bateman (C82),who have additionally presented the virtues of orthogonal acceleration of ions into the TOF region. Orthogonal acceleration in TOF is not itself new. Cotter and co-workers (C83) described in 1988 an instrument incorporating a pulsed LSIMS source, from which secondary ions were extracted into a TOF analyzer orthogonal to the axis of secondary ion ejection. Dawson and Guilhaus (C84)explicitly discussed the advantages of orthogonal acceleration in TOF MS, including those of controlling energy and spatial spreads in the TOF axial direction. In a sector/TOF combination incorporating orthogonal acceleration, now commercialized by VG Analytical/Fisons Instruments, precursor ions selected by a double-focusing analyzer are decelerated into a collision cell. Product ions and surviving precursors enter a drift region from which they are accelerated in an orthogonal direction. The need to float thecollision cell is dictated by the requirement that the range of drift velocities of product ions be not too broad, to enable sampling of the complete product ion population in a single extraction pulse ( 0 2 ) . Clearly, floating the collision cell reduces the collision energy achieved. For certain applications, such as the differentiation of leucine and isoleucine residues in peptides via examination of side-chain fragmentation, there is an established advantage to the use of high collision energies. Clayton and Bateman (C82)argued, on the basis of observations made by Martin (C85), that a laboratory frame-of-reference collision energy of 700 eV, using xenon as collision gas, is sufficient to yield peptide d-ions (via fragmentation of (a + 1) ions) from peptides as high in mass as 3000 Da. Thus, it was argued (C82),potential differences between ion source and collision cell of 1 kV are sufficient to access a full suite of diagnostically relevant fragmentations +
for large peptides. It remains to determine the extent of comparability between product ion spectra recorded on such sector/TOF tandem instruments and four-sector mass spectrometers, bearing in mind differences in the time scales of the two experiments. The incorporation of a reflectron TOF analyzer into the EB/TOF instrument described by Russell and co-workers (C81) allowed an alternative approach to tandem MS, compatible with a continuous ionization source (C86). A massselected precursor ion, which fragments in the field-free region between MS1 (EB analyzer) and the reflectron TOF, will yield a product ion and a neutral fragment, each of which retains the velocity of the precursor (ignoring kinetic energy release accompanying fragmentation). The neutral fragment will be unaffected by the retarding potential of the reflectron and will therefore impact the detector placed behind the reflectron, whereas the product ion will be reflected with a velocity equal to that with which it entered the reflectron. The residence time of ions in the reflectron is directly proportional to m / z so that the arrival time at the final detector, relative to the detection of the neutral, is a measure of m / z . This neutral/ion correlation approach (C86) is not limited in principle to pulsed ionization or injection techniques. Equivalently, the precise definition in time of pulsed ion formation is not required. The approach is, however, like all timecorrelation methods, dependent on a precursor ion beam of low current density, to minimize false neutral/ion correlations. Strobe1 et al. (C86) reported that inferior signal/noise ratios were obtained using their EB/TOF instrument when precursor ions were subjected to collisional activation, rather than when metastable decompositions were examined. The effect was attributed to increased neutral noise arising from neutralization of precursor ions by charge transfer with the target gas. These observations contribute to the continuing discussion on the importance (or otherwise) of charge-transferreactions in CAD. Statistical procedures have been applied to effect rejection of false neutral/ion correlations and improve signal/noise ratios (C87). If the original ionization event is precisely defined in time, then the time of detection of the neutral decomposition product allows the determination of the m / z ratio of the precursor ion, and neutral/ion correlations can be used to establish precursor/ product relationships using a single reflectron TOF instrument, albeit with a precursor ion resolution substantially inferior to that achievable with the EB/TOF configuration. In this instance the decompositions detected are any that occur spontaneously after ion acceleration, but prior to entry into the reflectron. This correlative technique was introduced some years ago by Della-Negra and Le Beyec (C88). Satisfactory resolution is achieved of product ions with m / z ratios (and therefore kinetic energies) close to that of the precursor, but lower mass products are poorly focused by virtue of poor penetration of the reflectron. A lowering of the reflectron voltage, and reexamination of the relevant portion of the spectrum, then achieves improved resolution of lower mass products (C89). Early and elegant use of the reflectron TOF instrument, with ion/neutral correlation techniques for determination of precursor/product ion relationships, was made by Standing and co-workers (C90, C91), who studied inter alia a rearrangement of metal-cationized peptides, which
results in the loss of the C-terminal amino acid residue with retention of a carboxyl oxygen (C90). This and related mechanisms were subsequently studied by several research groups using a variety of tandem MS approaches (C92-C94). A notable feature of a number of recent studies of peptides and small proteins using matrix-assisted laser desorption/ ionization has been the extent of metastable decomposition of protonated molecules after acceleration out of the ion source (see above). The sense of irritation that such decomposition can result in a lowering of the achievable mass resolution in a linear TOF analyzer, via kinetic energy release and a differential effect of fringe fields on precursor and product ions, has been replaced by the realization of the potential for structural analysis (C95). Spengler et al. ((242) reported prominent decay of peptide ions following matrix-assisted laser desorption, via losses of small neutrals corresponding principally to side-chain fragments. Three modes of instrumental analysis were employed in this exploratory study, viz. linear analyzer, short single-stage reflectron, and two-stage reflectron. The product ions of decay following acceleration were distinguished from stable ions by (i) differential behavior following minor variation in acceleration voltage, (ii) absence of separate signals due to metastable decay in the linear mode, and (iii) very small differences in flight times between metastable decomposition products and their precursors when the short single-stage reflectron was used. Interestingly, Spengler et al. (C42) emphasized the time scale of the experiment and pointed to the observations of Demirev et al. (C43) concerning the enhanced decomposition in plasma desorption TOF-RS and LSIMS-TOF of peptides when source residence times are extended. The analytical advantage of an extended time scale in .the examination of metastable decompositions is, of course, well recognized; e.g., Ngoka and Lebrilla (C96) demonstrated very high efficiencies of metastable decomposition of oligosaccharide ions during extended time-scale analyses using an FT-ICR instrument. Spengler, Kaufmann et al. (C97, C98) have more recently reported on the extensive sequence-specific fragmentation of peptides (to ca. 3000 Da) that may be observed when products of metastable decay are observed across a broad mass range, using stepping of the reflectron potential to achieve satisfactory focusing of product ions of widely differing kinetic energies. Gating of the ion beam between the ion source and the reflectron provides a crude means of precursor ion selection, with an effective precursor resolution estimated at 60 (C97). In principle, the precise location of the ion gate in the drift region is not expected to be important since, to a first approximation, precursor and product ions retain the same velocity. Despite the poor effective precursor-ion resolution, it is clear that this method provides a new approach to the definition of precursor/product relationships of analytical value. In particular, the high sensitivity achieved by the use of matrix-assisted laser desorption, together with the relatively high efficiencies of decomposition, makes this approach attractive for the analysis of samples of biological origin. Substantial effort is, however, required to characterize the gas-phase chemistry of ion activation and decomposition relevant to this technique. More generally, the relative merits of this and the various other experimental approaches now available for the study of precursor/product ion relationships Analytical Chemistry, Vol. 66,No. 12, June 15, 1994
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require a more substantive comparison than that provided thus far by Kaufmann et al. (C97). Martin and co-workers (C45) have made effective analytical use of the reflectron potential stepping technique (though without gating selection of the precursor ion) in their study of the extensive metastable decomposition of laser-desorbed protonated glycopeptides. The sequenceof the oligosaccharide component was determined from the full “postsource decay” spectrum assembled from the spectra recorded at various reflectron potentials. The same group used this experimental approach to study the facile gas-phase cleavage of AspXxx (particularly AspPro) bonds in protonated peptides (C99). Cornish and Cotter (CI00,C101) have reported on a new approach to the focusing of product ions of widely differing kinetic energies. Their design of a tandem TOF mass spectrometer, consisting of two serial reflectron TOF analyzers with an intermediate collision cell, now incorporates a curvedfield reflectron to allow the recording of product ion spectra without stepping or scanning of the reflectron potential (C100). The curved-field design replaced earlier versions of the tandem TOF instrument which incorporated two-stage (C80)or singlestage (C101)linear-field reflectrons. Early designs of tandem TOF instruments were reported by Schey et al. (C102, C103), whose area of interest was ion/surface collision phenomena. A tandem TOF mass spectrometer incorporating two linear analyzers, recently described by Jardine et al. (C104), was designed for FAB ionization with pulsed introduction of the secondary ions into the analyzer region. It is early days for development of tandem mass spectrometry based on TOF analyzers. However, it is already clear that single reflectron TOF instruments, which may become more common in combination with MALDI (see above), can also provide a cost-effective means of obtaining fragment ion spectra, though it is unclear as yet how the quality of such data and the universality of application of the method will compare with those of more established techniques. Similar remarks apply to the sector TOF hybrid instruments. One potential application for the latter is to on-line chromatography with full tandem mass spectrometry of all precursor ions as the corresponding compounds elute from the column. Such an experiment would take advantage of the very rapid repetition rate and high duty cycle of the TOF analyzer, but would place great demands on acquisition electronics and computer data logging. In this context, the remarkable development by Enke and co-workers (C105) of time-resolved ion momentum spectroscopy, in which fragment ions responsible for metastable peaks in a single magnetic sector instrument are unambiguously interpreted via their flight times following momentum analysis, has already achieved acquisition of the complete tandem mass spectrometry data field. Fourier TransformIon Cyclotron Resonance (FT-ICR)MS and Quadrupole Trap MS. FT-ICR was invented in 1974 by Comisarow and Marshall (C106-Cl08). The elegance of the original concept, and of subsequent developments by the original inventors and others (for general reviews, see refs C 109-C 112), lies in the close correspondence between a detailed mathematical theory of the phenomenon and the often ingenious experimental realizations of these mathematical constructs. The present review makes no pretense at at644R
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tempting to describe these developments, which have been extensively discussed in an admirable review by Marshall and Schweikhard (C112). Rather the utility of the technique in the context of analytical chemistry, both molecular structure determinations of unknown compounds and quantitative analyses of target analytes, will be examined in the spirit of the three introductory paragraphs of the present section. This discussion will thus ignore the enormous contributions made by both ICR and FT-ICR to the physical chemistry of ion/ molecule reactions. More recently, Marshall and co-workers (C113, CZ14) summarized their FT-ICR work devoted to highly accurate and precise (ppb level)determinations of mass differences between light isobaric elements, including that between 3He and 3H (tritium). If the latter mass difference can be determined to sufficiently high accuracy and precision, it will lead to a reliable value for the rest mass of the electron neutrino, a key quantity in cosmology which bears on the question of whether the universe will continue to expand indefinitely or eventually collapse back on itself. Of current mass spectrometric techniques, only FT-ICR has any hope of achieving this measurement, and then only as a consequence of the elegant physics and physical chemistry which have been devoted to development of the technique over the past 20 years. However, the present taskis different and must address the more routine question: to what extent does FT-ICR currently provide a useful analytical tool to the chemist and biochemist? In a scholarly 1991 review of what the authors referred to as “the teenage years” of FT-ICR mass spectrometry, Marshall and Grosshans (C111) examined the “behavioral flaws that were excusable in an infant” but which “soon became unacceptable for an adult” and described “the adolescent maturation of the FT-ICR technique”. In this admirable article (C111)the authors tooka hard lookat theexperimental technique which is the focus of their careers and faced up to the fact that, despite an exponential growth in the numbers of FT-ICR instruments installed worldwide (to 125 in mid1991 (CI 12)),they have not made much impact on the routine practice of analytical chemistry. An example, where the potential of FT-ICR for spectacular mass resolving power would have been expected to lead to its adoption as the standard technique, is provided by the requirements of the oil industry for high-resolution mass spectra of petroleum fractions in order to derive estimates of abundances of various compound classes. Sulfur compounds place particularly high demands on the resolving power; e.g., the aromatic CnH2n-16 series interferes with the CnH2,& series (C115). In these experiments, the vaporized oil components are introduced to the low-energy E1 source from an all-glass heated reservoir system via a molecular-flow leak and thus are not expected to place an inordinate burden upon the vacuum requirements of the FT-ICR cell. A random but nonscientific sampling of oil industry laboratories turned up no examples of FT-ICR instruments used for this particular purpose, one that seems tailor-made for one of the major strengths of the technique but which is still performed in practice by high-performance double-focusing sector instruments. Clearly this must reflect a “behavioral flaw“ of the kind referred to by Marshall and Grosshans (CZ 11). However, as emphasized by these authors, significant improvements in FT-ICR methodology have been
made over the last few years. Notable among these are the developmentof the stored waveform inverse Fourier transform (SWIFT) technique, which provides optimal excitation waveforms (C116, C117), improved understanding and control of signals at overtone and combination frequencies, and development of ion traps with highly uniform fields which enable reductions in limitations on mass accuracy, upper mass limits, selectivity of ion excitation or ejection, and precision of determinations of ion abundances (CIIl). In addition it has come to be recognized that, for many of the potential advantages of FT-ICR to be attained in practice, very low pressures Torr or less) must be maintained in the cell during the ion analysis. Accordingly, there has been a considerable effort in arrangements in which ions are formed in an ion source external to the ICR ion trap, with provision of ion guides for transmission of the ions from the source to the cell, and differential pumping of the two regions. Moving the ionization region outside of the magnetic field, where ions are trapped for subsequent analysis, thus avoids the problems arising from high pressures within the ICR cell, but at the expense of the magnetic mirror effect (CI 18), which inhibits ions, formed off-axis with respect to the symmetry axis of the magnetic field, from passing into the cell. The ion optical devices, which have been used successfully to permit introduction of externally generated ions into the high-field region, have been critically reviewed by Limbach et al. (C119). Most such devices use acceleration of the externally produced ion packet to overcome the magnetic mirror, with subsequent deceleration to allow efficient ion trapping in the ICR cell. The ion acceleration can result in a significant time-of-flight separation of ions of different m / z ratios, which can be advantageous as a means for discriminating against highabundance background ions but also precludes determinations of relative abundances (C119). Limbach et al. (C119) described the design and initial tests of an ion guide which does not require external acceleration lenses and thus no deceleration grids or lenses to facilitate ion trapping. Developments such as this should alleviate one of the more troublesome “behavioral problems” of adolescent FT-ICR mass spectrometry (CI 1I). Some remarkable successes in protein characterization, using external electrospray sources with FT-ICR mass analysis, have been reported from the groupsofMcLafferty (C120, C121),Laude (C122),andSmith (C59461, (2123, C124). However, it remains to be determined whether these successes represent heroic achievements of experiencedspecialists,routine results obtainable on demand using an accessible laboratory tool, or some intermediate situation. Somewhat similar considerations apply to the quadrupole ion trap mass spectrometer (ITMS), though this device has not been the subject of intense development for as long as the FT-ICR mass spectrometer. March has recently written a comprehensivereview (C125) which updates his excellent 1989 monograph (C126) on the subject. Other recent reviews by experts in the field include those by Todd (C127), by Cox et al. (C128), and by Schwartz and Jardine (B29). Some extraordinary achievements have been reported in the last few years. In 1991, a combined effort of research groups at Purdue, Finnigan MAT, and Los Alamos National Laboratory resulted in extension of the useable m / z range of the ITMS
to 70 000, with a mass resolution of 3000 (unit resolution at 50% valley at m / z 3000) ((2129). Also in 1991, an assault on the resolution attainable in ITMS was reported by three groups. Schwartz et al. (C130) reduced the radio frequency amplitude scan rate by a factor of 20 which, in combination with the axial modulation technique previously patented by Finnigan-MAT (C131), achieved a resolution of 33 000 at m / z 502 over a narrow scan range. Simultaneously, Goeringer et ai. (C132) achieved a resolution in excess of 45 000 for the same m / z 502 ion by scanning the resonance ejection frequency. Even these achievements were overshadowed by the astonishing result reported by the Purdue group (C133), who achieved a resolution of 1.13 X lo6 for the CsI cluster ion at m / z 35 10, using a 2000-fold decrease in scan rate from the usual value of 5555 m / z units/s. In 1993 Londry et al. (C134) demonstrated a resolution of 1.2 X lo7 for m / z 614 at a scan rate of 0.1 m / z units/s, and demonstrated the dependence of resolution on scan rate over more than 5 orders of magnitude for the latter. At scan rates lower than 0.1 m / z units/s the resolution deteriorated slightly, but above this value peak width increased almost linearly with scan rate. These data (C134) were compared with theoretical predictions of Goeringer et al. (C135) and shown to correspond to the predicted curves but shifted upward on the scan-rate axis by about 2 orders of magnitude. Extension of these studies to ions covering the range m / z 69-6 14has been reported, together with a brief description of an approach designed to improve the accuracy of mass assignment (C136). Admiration for these remarkable achievements, however, is no substitute for the hardnosed assessment outlined in the introductory paragraphs of this section. Current limitations of the highresolution mode for ITMS include the slow scan speeds and correspondingly narrow mass ranges, the drifts of peak positions which make scan averaging disadvantageous and mass assignment difficult, and susceptibility of trapped ions to CAD via collisions with the helium buffer gas at the long trapping times required (C134). Cooks et al. (C137)have pointed out that another area of ITMS needing improvement is the dynamic range, where adaptation of the SWIFT technique from FT-ICR (see above) is improvingperformance. McLuckey (CI 38) has summarized progress in applying ITMS to biological problems. Considerable effort has also been devoted to the ITMS as a “tandem-in-time” tandem mass spectrometer, capable of investigating several stages of fragmentation, as discussed by McLuckey et al. (C139). More recently, Glish (C140) has discussed the role of the ITMS in environmental chemistry, which depends heavily on its applicability as a chromatographic detector. The use of a simple ITMS as a mass-selective detector for gas chromatography is well established and commercially available, but extension to other chromatographies, and to on-line tandem mass spectrometry, is not straightforward. The low energies accessed in CAD in ITMS imply that only one or two dissociation channels are accessed, but this disadvantage can be overcome by exploiting the multiple-stage reaction sequenceswhich areaccessible (C139). However, a practical difficulty is that the resonant frequencies of the analyte ions (selected precursors) depend on the total number of ions in the trap, as a result of ionlion interactions which also limit the number of ions which can be stored at Analjltcal Chemistry, Vol. 66,No. 12, June 15, 1994
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any one time and thus the dynamic range (C140). This presents a serious problem for LC/ITMS experiments using electrospray ionization, for example, in which the mobile phase can provide a high background of low-mass ions. Ejection of this low-mass background by resonant ejection is possible but runs the risk of losing important analyte ions since the m / z values of unknown analytes are not known in advance. By the same token, resonant excitation of unknown analyte ions for tandem mass spectrometry requires foreknowledge of the resonant frequencies, which are not necessarily known accurately even if the desired m / z values have been determined, as a result of the space-charge problem (C140). The use of random noise applied to the ITMS end caps as an activating agent has been described by McLuckey et al. (C141) as an effective means for the CAD of trapped ions, independent of the m / z ratio and the number of ions in the trap. Despite some disadvantages described by the authors (C141), this approach holds promise for the implementation of ITMS as an on-line tandem mass spectrometer for chromatography. McLuckey et al. (C142) have also described a CE/ITMS experiment using electrospray ionization, in which the random noise excitation method was combined with resonance ejection of background ions that were resistant to CAD to successfully improve the dynamic range of detection. Schwartz and Jardine (C143) have demonstrated that the same techniques as those used to generate high-resolution mass spectra can be adapted to selection or storage of a very narrow m / z window with high efficiency. Clearly, some clever and imaginative solutions have been devised to the problems remaining for development of ITMS as a widely applicable analytical tool. At present it appears that many of the achievements summarized above are not available on demand and remain the preserve of the specialist groups who were responsible for them. Surface-Induced Dissociation. Collisions of polyatomic ions with gaseous targets, as a means of ion activation to promote structurally informative fragmentations, is well established. The first attempt tosubstitute a solid for a gaseous target, as an activation method for tandem mass spectrometry, was reported in 1975by Cooks et al. (C144). Surface-induced dissociation (SID) has been the subject of considerable developmentsince then, mostly in Cooks' laboratory at Purdue. The fundamental principles and applications of SID were extensively reviewed by Cooks et al. (C145) in 1990. The advantages of SID over gas target CAD include the ease with which large amounts of energy can be transferred to the ion, with a remarkably narrow distribution and a strong dependence on collision energy, which can thus be tuned as desired, and the reproducibility of the SID spectra. Disadvantages of SID (C145)include the disappointing efficiency due mainly to the competition from neutralizing collisions, the poor ion optical quality of the emerging ion beams, uncertainties in the nature of the collision surface when installed in a mass spectrometer vacuum system, and the nonlinear optical path used in most experimental arrangements thus far. Considerable development of the technique, in a variety of instrument configurations, has been reported since the date of the major review (C145). Wysocki and co-workers ((2146) compared the relative merits of three tandem quadrupole configurations. Two quadrupoles were arranged (i) in-line with an on-axis conical surface, (ii) in-line with an off-axis 646R
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surface, and (iii) orthogonally with a surface placed to intersect the axes of the two quadrupoles. A comparison of the relative energy transfer and transmissionefficienciessuggested (CI46) that the 90' arrangement is preferable to the in-line devices examined. SID has also been studied in four-sector instruments by Jennings et al. (C147, C148), with respect to the effects of collision energy and the nature of the surface. The dissociation of the molecular ion of fluorobenzeneafter collision with Teflon and stainless steel targets suggested that the fluorinated target was the most efficient for promoting dissociation,although it was confirmed that the energy-transfer distribution is considerably more narrow for SID than for CAD using gaseous targets. Other work on SID of protonated peptides generated by LSIMS, in a four-sector mass spectrometer (CI49),has shown a 7.2%dissociation efficiency for 80-eV collisionsof protonated leucine enkephalin with a brass off-axis target. A hybrid instrument for the study of molecular dissociations after collisions with surfaces was reported by Li et al. (CIS@,in whicha BE-surfaceWB tandemarrangement (B, magnetic sector; E, electric sector; W, Wien filter) was used with a microchannel plate used to effect SID. The molecular ion of pyrene was used as a precursor, and differences in fragmentation were noted to increase with increased collision energy. However, the resolution of the product ions was low (z200) (C150). Considerable attention has recently been paid to the nature of the target surfaces used in SID. Early studies used untreated metal surfaces that were noted to be covered with hydrocarbon adsorbates from the vacuum system (C145). The use of self-assembledalkanethiol monolayer target surfaces on a gold substrate was reported independently by two groups (C151, C152),and the use of these surfaces has been pursued in both applications (fullerenes (CI 53) and peptides (CI 5 4 ) ) and fundamental studies (C155). An additional feature of ion collisions with surfaces which have been chemically prepared is the formation of reaction products in which functionalities from the target surface are incorporated into the precursor ion. For example, reactions at fluorinated surfaces have been documented to lead to inclusion of fluorine and of CF3 (CI 56, C157). This trend toward more fundamental studies of the ion/surface interactions is epitomized by the sophisticated custom-built instrument described by Cooks et al. (C158). This BE-surface-EQ instrument is capable of studying scattered ions as a function of both angles of incidence and of extraction, and the energies of both pre- and postcollision ions can be measured. SID has also been examined (C159) using a surface composed of a liquid perfluorinated polyether with a room temperature vapor pressure of 3 X Torr. The SID behavior was found (C259) to be similar to that induced by a fluorinated self-assembled monolayer, but the ease of preparation and stability of the surface were superior. Zare et al. (C160) have described studies of the efficiency of energy conversion for SID using a tandem time-of-flight instrument with a stainless steel surface. SID has also been used in a time-of-flight analyzer to fragment large ions and thus increase the detection efficiency of the system without the need to apply high postacceleration voltages (C161).SID processes have also been examined in quadrupole ion traps (C162) by the use of a fast dc pulse to accelerate the trapped ions into the surface of the end caps of the ion trap.
Again, SID has been the subject of some commendably imaginative work, and certainly an alternative to gas target CAD would be desirable. However, at present it seems doubtful that this technique is ready for use as a routine analytical tool. Mass Spectrometric Studies of Noncovalent Interactions. Ions incorporating noncovalentlybound entities are frequently observed using a variety of mass spectrometric techniques. Thus, for example, matrix-derived multimers are commonly used in LSIMS to establish or confirm mass assignments and the study of cluster ions represents an extensive area of independent enquiry (CI 63). Observation of the decomposition of proton-bound heterodimers is the basis for the “kinetic method” for the determination of relative gas-phase proton affinities (C164). The recent burgeoning of interest in noncovalently bound ions, however, is largely attributable to the observation of such gas-phase species derived from biomolecules and the supposition that these intermolecular interactions reflect condensed-phase properties of biological significance. Most such studies have involved the use of electrospray ionization. Limited studies using matrix-assisted laser desorption have suggested that, with this technique, caution is required in interpreting the observation of gasphase multimeric ions as a true reflection of condensed-phase interactions (CI65,C166). It is generally accepted that the electrospray process initially yields highly solvated ions in the API source which are subsequentlydesolvated,by collisionor heating in the interface, to yield the “naked” ionic species which is the normal target of analysis. A perspective on these processes has recently appeared (CI 6 7 ) . Clearly, the observation of noncovalently bound species such as protein/carbohydrate pairs, in the electrospray spectrum, implies a more avid association than between solvent (typically water) and macromolecular ions. In addition to the studies of noncovalent intermolecular interactions (reviewed in moredetail below), electrospray mass spectra of proteins have been interpreted in terms of the effects of intramolecular associations which determine protein conformation (CI 6 8 4 1 72). Thus, the distribution of charge states has been interpreted to reflect the “tightness” of protein folding, with higher charge states suggesting a more open conformation (CI 72). While the proposition that the gasphase charge-state distribution is an indication of condensedphase tertiary structure cannot necessarily be assumed to be valid, the “titration”of condensed-phase composition and gasphase charge state has provided convincing evidence that (in some cases at least) a shift in charge-state distribution of electrosprayed ions coincides with conformational change in solution (CI 72, CI 73). The shift in charge-statedistribution of electrosprayed ions has also been used to monitor the heatinduced denaturation of proteins (C169, C174). Auxiliary techniques have been employed to probe protein conformation. Thus, the rates and extents of hydrogenldeuterium exchange in proteins, as determined by electrospray MS, have been interpreted as reflecting protein conformation (CI 70, CI 75CI 78). A particularly elegant example of the latter approach is the work of Miranker et al. (C179), who have pointed out the complementary value of electrospray MS and of NMR in studies of protein folding using deuterium exchange. Thus, NMR allows the determination of average isotope incorpora-
tion at individual sites. In contrast, electrospray MS does not permit estimation of 2Hexchange in a residue-specificmanner, but does permit determination of different populations of protein molecules with different masses (C179). Additional strategies have been adopted to probe protein conformation in the gas phase, as opposed to interpreting gas-phase properties as a reflection of condensed-phase states. Introduction of multiply charged proteins into the gas-filled rf-only quadrupole of a triple quadrupole instrument has resulted in selective depletion of species with larger cross sections, representing (C180) more open conformations. A method for the determination of cross sections of protein ions, based on energy loss on passage through a collision gas, has beendescribed by Covey and Douglas (C181). A particularly intriguing study has been reported by McLafferty and coworkers (CI 7 0 ) , who determined the extents and rates of deuterium exchange between multiply charged protein ions and 2H20in the gasphase using a FT-ICR mass spectrometer. For cytochrome c, at least three distinct conformers were suggested by differing extents of *H exchange. Interconversion between the conformers appeared to be extremely slow in the gas phase, perhaps suggesting that interconversion between conformations in the condensed phase may be facilitated by solvent disruption of intramolecular associations. Multimeric forms of peptides (CI82) and proteins (C183) have been observed by electrospray ionization,with appropriate attention to the choice of solvent and interface conditions. Thus, for example, the dimer of y-interferon was observed when the electrospray solvent was distilled water at a measured pH of 6.7, while electrospray from aqueous methanol yielded only the monomer (C184). Multimeric forms of concanavalin A have been studied using an extended mlz-range quadrupole (CI85)and using a magnetic sector instrument (C171). Observation of monomeric, dimeric, and tetrameric, but not trimeric, forms suggested specific, rather than nonspecific, interaction and was in accord with solution studies (CI71). Dissociation of the multimeric forms could be achieved by increasing the skimmer potential in the electrospray interface to achieve more energetic collisions of the electrosprayed ions (CI 71) . The merits of extended m/z range detection of electrosprayed ions have been presented by Smith and co-workers (C186), who argue that one advantage is the possibility of detection of otherwise “Coulombically labile” noncovalent interactions. The use by this group of an extended-range (m/z 45 000) quadrupole instrument has demonstrated that high m/z ions are indeed formed under appropriate conditions; e.g., singly deprotonated bovine pepsin was detected at m/z 33 750. These observations raise interesting questions concerning the mechanism of ion formation in electrospray (the ion evaporation model vs the charged residue model) and the relationship (if any) between the charge state in solution and in the gas phase (C186-CI88). These mechanistic uncertainties suggest that the study of (inter alia) multimeric protein ions and other noncovalent interactions remains largely empirical. Detection by electrospray of specific interactions between dissimilar and functionally related molecules represents an area of particular interest and biochemical relevance. Katta and Chait (C189) demonstrated the detection of the intact Analytical Chemistty, Vol. 66, No. 12, June 15, 1994
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heme-globin complex in native myoglobin. Collision-induced dissociation in the interface or in the second quadrupole of a triple quadrupole instrument may be used to distinguish noncovalently bound heme in myoglobin and hemoglobin from the covalently bound heme prosthetic group in cytochrome c (CI 90). Ganem et al. were successful in observing complexes of enzyme-substrate and enzyme-product (C191) and in detecting noncovalent receptor-ligand complexes (C192). Further examples of the detection of protein-ligand complexes include the occupied cobalamin-binding domain of methionine synthase (CI 93), a ternary complex between the dimeric enzyme HIV- 1 protease and a substrate-based inhibitor (C194),and the interaction of cytidylic acids with ribonuclease A (C195). Noncovalent inclusion of small peptides within a cyclic oligosaccharide structure (0-cyclodextrin) has also been detected by electrospray MS (C196). Looet al. (C197) have reported the electrospray M S analysis of heterodimers comprising angiotensin I1 and complementary 'antisense" analogues (meaning the sequences encoded by DNA complementary to that coding for angiotensin 11). The relative abundances of the heterodimers broadly correlated with the extent of interaction in solution, as judged by NMR spectroscopy (C197). Theobservation of heterodimericions (albeit of low abundance), where N M R suggested the absence of condensed-phase interaction, however, may be interpreted either as a superior sensitivityof the electrospray MS approach or as a result of the formation of dimeric species during gaseous ion formation. Recent reports have described the detection by electrospray MS of intact oligonucleotide duplexes (C198, C199). In a limited study (C198) it was demonstrated that a duplex consisting of A-T interactions survived the conditions of ion formation less successfully than duplexes comprising extensive C-G pairing, consistent with the presenceof only two hydrogen bonds in each A-T interaction. Low-energy CAD readily dissociated the duplex ion (C198). A DNA quadruplex consisting of (CGCG4GCG)4 was observed by electrospray MS in the presence of sodium cations, consistent with observations of the formation of a quadruplex in solution (C200). Interactions between regulatory guanine nucleotides and ras-proteins have been studied (C184, C201, C202). Detection and differentiation of the GDP- and GTP-bound forms by electrospray MS is of biochemical interest in view of the differing activities of the two forms. The multiplicity of reports of successful detection of noncovalent complexes by electrospray mass spectrometry should not lead to the conclusion that the failure to detect such a complex necessarily implies its absence in the condensed phase. As noted above, one design objective of the electrospray interface is the destruction of some noncovalent interactions (desolvation), and it is clear that the preservation of other noncovalent interactions into the gas phase is highly dependent on the selectionof sample introduction and interface conditions (C167, C203). The present limited understanding of the mechanisms of ion production, and of the processes occurring in the interface region, suggests the need for continued caution in interpreting both qualitative and quantitative data on these noncovalently bound species. Collision-Induced Light Emission. The UV/visible light emitted by polyatomic cations, collisionally activated at 648R
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kiloelectronvolt energies in a commercial analytical mass spectrometer, has been studied recently by Holmes et al. (C204, C205). In these first reports, cutoff filters were used to obtain crude histograms of the intensity of the emitted radiation as a function of wavelength in the range 180-680 nm. The collision-induced emission spectra of cations of the compositions (C2,H4,O)'+ and (Cz,H5,Cl)'+ proved to be isomer characteristic and were observed to change with the nature of the collision gas and with the time window for observation (C204, C205). A scanning grating monochromator has now been installed to replace the filters, permitting emission spectra of higher resolution to be obtained (C206). Studies of simple diatomic and triatomic projectile ions, with a number of target gases, have yielded emission spectra due to electronic transitions in the projectile ions and in neutral molecules formed from them by electron capture from the target gas. The importance of electron capture as a preliminary to emission in collisions at similar energies has been shown previously in studies on simple systems (monatomic and diatomic species only) by Leventhal (C207). Emissions arising from neutral and ion fragments, as well as from ionized target species, have also been observed (C206). Results for the ions N2*+,02*+, and C02'+ have been submitted for publication (C206) and are in accord with previous studies using specialized apparatus. Most recently, high-resolution emission spectra arising from collisionsof polyatomic projectile ions such as the (CzlH4,0)*+ and (C2,H5,Cl)'+ isomers, and from the benzene radical cation, have been obtained (C206). These latter emissions appear to arise predominantly from excited-state fragments, particularly atomic and diatomic ions and radicals. These elegant studies (C204-C206) are unlikely to lead directly to useful analytical techniques, but are of great importance for the understanding of the collisional activation process. Apart from the unparalleled detail thus provided, the integrated intensity of the emission (extremely low) is important for the energy balance describing collisionalactivation events (C2O8). In this context, the considerations initiated by Dunbar et al. ( C 2 0 9 4 2 1 2 ) of infrared emission from ions suggest that this latter phenomenon is probably unimportant for tandem-in-space instruments, but that the time scales are more nearly matched for trapped ions in, for example, FT-ICR experiments. Such a deactivation mechanism has been invoked recently by Castro and Wilkins (C50)to help explain the improved resolving power obtainable for large laser-desorbed ions when a light-absorbing matrix is used.
D. MASS SPECTROMETRIC DETECTION FOR ON-LINE HIGH RESOLUTION SEPARATIONS A measure of the growing importance of this aspect of mass spectrometry is the large number of reviews on the subject which have appeared over the last few years. Some of these are listed in the 1992 Analytical Chemistry biennial reviews of the various separation techniques (01-06). More recent reviews are mentioned at the appropriate points below. The correspondingly very large volume of recent publications, describing work in which a mass spectrometer was used as the on-line detector for a separatory technique, makes it necessary to be highly selective here. The publications cited in this section were chosen on the basis of their contributions to understanding the physicochemical basis for a particular
technique or as examples of development of new techniques or significant extensions of well-established ones. Even here limitations of space have meant that the choice has had to be somewhat arbitrary. Many of the applications of X/MS techniques, where X denotes a separatory technique such as LC, SFC, CE, etc., are covered in later sections of this review. This review naturally views the X/MS techniques primarily from the MS viewpoint. To the chromatographer, however, the mass spectrometer is just another detector (07, OB),whose characteristicsmust be weighed against those of rival detectors. Such comparisons are particularly important for quantitative analyses (some formal considerations for use of internal and external standards in conjunction with MS detection have been published (D9)). In this context, the following characteristics and figures of merit are of interest (these are not all mutually independent): (i) the nature of the detector as destructive or nondestructive; (ii) the degree to which the detector response can be classified as dependent on the mass flux or on the concentration of the analyte in the eluate; (iii) the degree of postcolumn band broadening introduced by the detector, including its transfer line, stagnant volume, and detection volume; (iv) the degree of electronic broadening of the observed chromatograms, arising from slow detector rise times; (v) the degree of selectivity of the detector, i.e., the chemical information content of the detector signal; (vi) the degree of universality of the detector; (vii) the dynamic range and linear range of the detector, under specified chromatographic conditions; (viii) the sensitivity (response), Le., change in signal (peak area or height) per unit change in analyte mass flux or concentration, under the specified chromatographic conditions; (ix) the detection limit (mass or concentration), usually determined in terms of peak height (not area) relative to the intrinsic short-term electrical noise output of the detector; and (x) other considerations, such as compatibility with buffers and other mobile-phase modifiers. All mass spectrometers are destructive detectors (i), to the extent that at least the portion of sample introduced to the ion source cannot be returned to its owner. (This emphasizes the important distinction between the minimum quantity of sample consumed by the instrument to give a meaningful response and that necessary to fulfill requirements of sample handling, injection, etc., to achieve the same end.) With respect to (ii), E1 and CI ionization sources are classic examples of massflux-dependent detectors (if the sample flow rate is reduced to zero, so is the response), while the archetypal concentrationdependent detector is the UV/visible absorption (BeerLambert law) detector (if the analyte flow is stopped thesignal remains constant). Less drastic decreases in sample flow rate lead to corresponding decreases in the chromatographic peak height but leave the integrated peak area fixed for the massflux-dependent detector, while for the true concentrationdependent detector the peak height is essentially unchanged and the peak area is increased (D8).(This comparison clearly involves a fixed quantity of analyte in a fixed injection volume, under circumstances where changes of flow rate do not significantly alter the linear length of the "plug" of analyte solution passing through the detector.) Among other things this distinction emphasizes that figures of merit which measure sensitivity need to be carefully defined (e.g., peak area or height) and are properties of the combined X/MS system. It
might be thought that all mass spectrometer ionization sources show mass-flux dependence, since halting the flow of analyte always reduces the M S signal to zero. However, important examples exist (see below) where some ionization sources display some of the characteristics of a concentrationdependent detector. It is unusual for an MS detector to give rise to peak broadening under criterion iii or iv, provided that appropriate precautions are taken with respect to plumbing and to MS scan speed relative tochromatographic peak widths. The latter condition cannot always be fulfilled, particularly for some separatory techniques which give rise to very narrow peaks, so that the mass analyzer then becomes the limiting factor rather than the ion source. Criteria v and vi are sometimes regarded as mutually exclusive; e.g., the use of immunochemical detection (DIO)achieves extremely high selectivity by virtue of its narrow applicability (limited crossreactivity). However, the information content of a mass spectrum (or MS/MS spectrum in the case of soft ionization techniques) is sufficiently high that MS detection can simultaneously provide high degrees of both selectivity and universality. In this regard it is pertinent to note that the FT-IR spectrometer is approaching MS as a useful detector combining characteristics v and vi, now that techniques of cryogenic deposition of chromatographic eluent have greatly alleviated the sensitivity problem (DII). The figures of merit summarized in criteria vii-x vary widely with the nature of the analyte and other parameters and must be determined on a case-by-case basis. The preceding general discussion is intended to set the stage for what follows. It is important to note, however, that it represents a considerableoversimplification of the problems facing the analyst dealing with real-world (i.e., chemically complex) samples. Many instrumental parameters interact with one another in complex ways. For example, use of microbore LC columns (e.g., 0.32-mm i.d.) implies eluate flow rates in the low microliter per minute range, compatible with MS ionization techniques such as continuous-flow FAB and electrospray (see below) without need for a postcolumn split. However, there are practical difficulties such as those involved in obtaining good submicroliter injectors and in generating reliable gradient elution conditions at the low flow rates. Perhaps the most severe of these practical problems derives from the increasing difficulty in packing columns uniformly as the inner diameter is decreased. As a consequence, the efficiency of a microbore column (number of theoretical plates per unit length) can be considerably less than that of a standard analytical column (2.1- or 4.6-mm i.d.) packed with the same stationary phase. On the other hand, the great advantage of using a microbore LC column with a low flow rate M S technique lies in its sample utilization efficiency. For example, assuming that columns with equal numbers of theoretical plates are compared, each operated at its optimum flow rate, the analyte concentrations at the peak crests will be exactly the same if the injection volumes are proportional to the cross-sectional areas (typically 25,5.2,1.2 and 0.12 pL for column inner diameters of 4.6, 2.1, 1.O and 0.32 mm, respectively). For a scarce sample this can be an overriding consideration, particularly if a postcolumn split is required for reasons of compatibility with the MS. However, the greater sample loadings permitted with columns of larger AnaWiicel Chemistty, Vol. 86, No. 12, June 15, 1994
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inner diameter may be important for characterizationof minor componentsin a complex matrix. There is no general rule for determining the best compromise, which must be approached in the context of the particular analytical problem and of the instrumental resources available. The following discussion of recent developments is organized as two different cross sections of this complex area of X/MS analyses. The first cross section considers the various ionization sources and associated interfaces which permit coupling of liquid or gaseous chromatographic eluates to the MS vacuum system. The second cross section displays the role of the MS as on-line detector for the range of separatory techniques in current use. The first cross section will contain most of the relevant fundamental physical chemistry research while the second is more concerned with practical devices and techniques, though the distinction becomes blurred in some cases. Ionization Sources and Interfaces for Ms as a Detector. It is no accident that mass spectrometry was first used as a detector for gas chromatography, since compounds with sufficient volatility and thermal stability to survive GC separation are well suited to MS analysis by classical E1 and CI techniques which require vaporization prior to ionization. It is relatively easy to arrange for MS vacuum pumps to deal with the gaseous mobile phase, and GC/MS is now a fully mature technique. The fundamental problem for compounds which are involatile and/or thermally labile, whose separation requires HPLC or other liquid-phase techniques, has always been to transform them from dilute solution at ambient temperatures to gaseous ions in the MS vacuum chamber. Those LC/MS interfaces designed for use with the same E1 and CI sources as are employed with GC/MS rely on rapid heating to enhance volatilization over pyrolysis, as discussed some years ago by Beuhler et al. (012)and by Daves (013). This approach (012, 013) relies on a higher Arrhenius activation energy for vaporization (breaking intermolecular bonds) than for pyrolysis (breaking intramolecular bonds, but partially compensated energetically by formation of new bonds). For such a case where the rate of pyrolysis exceeds that of vaporization at lower temperatures, there must be a crossover temperature at which this relationship is reversed. Rapid heating through this crossover point brings the sample to a condition where vaporization is favored without having to undergo extensive pyrolysis during the heating process. This process is exploited, for example, by direct-exposure probes for batch analyses in which the sample is deposited on a thin metal wire whose temperature can be raised at very rapid rates. The moving-belt (MB) LC/MS interface (DI4),another mature technique which has been extensively reviewed (D15), relies on a relatively rapid heating rate, at the point where the belt approaches or enters the E1 or CI source, to enhance volatilization with moderate success. Although developed for LC/MS applications ( 0 1 4 ,the MB interface is sometimes used as an alternative to the direct solids probe for batch samples to be analyzed by E1 or CI. Although the sample temperature cannot be controlled as well as in a direct solids probe, the relatively rapid rate of heating often permits useful E1or CI spectra to beobtained through use of the MB interface in cases where other sample introduction techniques fail. As 650R
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an LC/MS interface the MB is, however, mainly restricted to analytes of appreciable volatility and thermal stability. For example, it was possible to analyze polycyclic aromatic compounds with molecular masses up to 580 Da by LC/MS using a MB interface coupled to an E1 source (016). The flash-heatingeffect may also contribute to the success of the particle-beam LC/MS interfaces, which can deal with large flow rates (1 mL/min) under gradient elutionconditions, and yield library-searchable E1 spectra in suitable cases. This point is also made in an excellent recent review (017)of the history, development, and applications of particle-beam LC/ MS since its introductionin 1984 by Willoughbyand Browner (018). This timely review (017) emphasizes not only the degree of universality of this detector, compared with most other LC/MS interfaces, but also that sometimes severe problems of sensitivity, band broadening, and nonlinear response remain as serious limitations. The “carrier effect” is particularly puzzling, as exemplified by observations made in the course of quantitative analysis by isotope-dilution particle-beam LC/MS (019,020). Recent developments of the particle-beam interface have included its combination with liquid-assistedsecondary ion mass spectrometry of the particles after deposition on a target. Kirk and Browner (021)used a matrix initially deposited on the target prior to insertion, while Sanders (022)included matrix in the sample solution (mobile phase) so that each individual particle contained sufficient matrix. This work (021,022) was motivated by a desire to use the full eluent from 4.6-mm4.d. columns, possibly with gradient elution, since continuous-flow LSIMS (discussed below) is limited to flow rates of 5 pL/min or so. More recently (023,024)surface ionization techniques have been investigated as possible combinations with the particlebeam interface. Useful results were obtained in these preliminary experiments. Another physical effect, which probably contributes to the success of some LC/MS interfaces in providing useful E1 spectra of compounds for which pyrolysis dominates vaporization in a standard insertion probe, is that of the increase in equilibrium vapor pressure over that of the bulk substance as the particle size is decreased. This effect reflects the imbalanceof intermolecularforces on molecules in the surface layer of particles sufficiently small that the surface/volume ratio is large. In practical terms, the advantage is common to all devices in which the liquid eluent is nebulized into fine mist particles, from which solvent (mobile phase) is preferentially evaporated to leave extremely small particles of solute (analyte) containing only a few molecules. The relationship between the vapor pressure P above a curved convex surface of radius r and its value POabove the bulk material is given by the Kelvin equation (025): In(P/Po) = 2My/RT dr where y is the surface energy per unit area (reflecting the intermolecular forces), M the molar mass, and d the density. Evaluation of this equation shows that, for liquids, the effect is not appreciable until r falls below about 1odcm (0.01 pm). However, if nebulization of the LC eluent initially produces micrometer-sized droplets, the dry solute particles remaining after solvent evaporation are expected to be no larger than
10“ cm for initial solution concentrations typical of LC/MS experiments. Moreover, the surface energies y of solids are appreciably larger than those of the corresponding liquids, so an appreciable increase of P over POshould result. It is likely that, in most LC/MS interfaces, the rates of solute volatilization are at least as important as the equilibrium vapor pressures discussed above. However, if expressed in terms of activated complex theory, for example, these rates will vary with the same parameters as those accounted for in the Kelvin equation. The increase in ease of solute vaporization due to prenebulization of the LC eluent probably contributes to the success of both the particle-beam interface and the heated pneumatic nebulizer (HPN) interface combined with atmospheric pressure chemical ionization (APCI). Gentle heating (100-1 20 “C) at atmospheric pressure facilitates evaporation of solvent from the nebulized droplets. The HPN approach to coupling LC with MS was introduced by Thomson et al. ( 0 2 6 ) , building on earlier work by Henion et al. 1027) and by Horning et al. ( 0 2 8 ) ,which did not, however, incorporate prenebulization of the LC eluate. The HPN interface is a deceptively simple device ( 0 2 9 , 0 3 0 )which has proved to be remarkably useful in high-throughput quantitative analyses of analytes of moderate molecular masses and low to moderate polarities. For example Kaye et al. ( 0 3 1 ) found that quantitative analysis of abanoquil in blood samples, using an LC/MS/MS technique incorporating a HPN interface with APCI, was limited by the sample cleanup procedure to 50 samples per day, although the LC/MS/MS instrument itself analyzed this number of samples in 2.5 h. Excellent precision and accuracy were obtained ( 0 3 1 ) . Another recent example of a real-world problem, in which the HPN interface with APCI was found to provide the best performance of all the LC/MS interfaces tried (HPN, ion spray, thermospray, and particle beam, in decreasing order of performance for this example), is provided by N-methyl carbamate pesticides in vegetables ( 0 3 2 ) . A similar study ( 0 3 3 )proved thesuitability of this approach for multiresidue pesticide analysis for a wide range of chemical classes. LC/MS quantitation of a renin inhibitor in serum, over the concentration range 50 pg mL-’ to 10 ng mL-I, was demonstrated by Fouda et al. ( 0 3 4 ) using a HPN-APCI method. Over 4000 clinical samples were thus analyzed with excellent precision with the aid of an internal standard. It is also possible to tailor the APCI plasma to the analyte of interest by controlled doping. For example, LC/MS analysis of fullerenes and related compounds was achieved ( 0 3 5 ) using a HPN interface with postcolumn addition of benzene, so that charge-transfer ionization by benzene molecular radical cations was the dominant ionization mechanism in the APCI plasma. Many more examples could be cited in which the HPN interface has proven sufficiently rugged for unattended overnight operation with an autosampIer, providing quantitative data of excellent accuracy and precision. It is compatible with full eluate flow (no split) from 4.6-mm columns operated under gradient elution conditions, and involatile buffers in the mobile phase are swept away by the rapid ventilation in the APCI source. Although it does not usually receive the attention paid to the spray techniques discussed below, the HPN interface has already proven itself to be a reliable workhorse for analytes covering a useful range of molecular
mass and polarity ( 0 3 0 ) . In many respects the HPN-APCI combination and the electrospray/ion spray techniques (see below) complement one another very well. For convenience, the problems associated with transferring the analyte-derived ions from the atmospheric pressure source to the MS vacuum will be discussed below in connection with the electrospray technique. All of the LC/MS interfaces discussed thus far have addressed the problem of the large volume of liquid mobile phase by selectively removing the more volatile solvent before the vaporized analyte reaches the ionization region. All of the remaining interfaces exploit the mobile phase in some way in order to produce gaseous analyte ions. Conceptually the simplest, and chronologically the first, is the direct liquid introduction (DLI) interface in which a flow of about 10 pL/ min is introduced directly into a conventional CI source, where the solvent acts as the reagent gas. The DLI method was first introduced over 20 years ago ( 0 3 6 , 0 3 7 )and has been reviewed recently ( 0 3 8 ) . Some means of nebulization is usually incorporated, in view of the low rates of heat transfer to the liquid droplets under the low-pressure conditions of the CI source. This limitation, which does not apply to the atmospheric pressure conditions pertinent to the HPN interface, contributes to the irregular response often observed with DLI interfaces. One method of alleviating this problem is to preheat the liquid as it flows through the DLI capillary prior to entering the CI source ( 0 3 9 ) , an approach which starts to resemble the thermospray technique discussed below. A more recent version of the “preheated DLI” method uses a conventional GC/MS interface as the preheater ( 0 4 0 ) . The DLI interface is subject to clogging problems and is incompatible with involatile buffers, but is inexpensive and easy to implement if no specialized LC/MS instrumentation is available. The history of the development of the thermospray (TS) LC/MS interface, by Vestal and his collaborators, has been well described by its inventor ( 0 4 1 ) and also in excellent reviews by Arpino ( 0 4 2 , 0 4 3 ) . Typically, the flowing solution, which is most commonly aqueous and thus confined to reversedphase LC, is pumped through a strongly heated capillary so that some of the solvent is vaporized and expands out of the capillary tip at sonic velocities, thus nebulizing the remaining liquid. The fast beam of vapor and mist particles emerges on an axis perpendicular to the main optical axis of the MS, and ions are extracted into the analyzer via a sampling cone. When a volatile salt is included in the liquid flow, typically 0.1 mol/L ammonium acetate, analyte-related ions are formed even in the absence of any additional ionizing agents. This “filamentoff“ operating mode is an intriguing feature of TS, which has given rise to much speculation concerning the mechanism(s) responsible. The currently accepted explanation is that, at the high temperatures used (200-300 “C), the ammonium acetate yields gaseous ammonium and acetate ions which act as efficient CI reagent ions ( 0 4 4 ) . In some cases ionization is enhanced by using an auxiliary electron beam (“filamenton mode”) or an electrical discharge (“Plasmaspray” in the terminology of one instrument manufacturer). When applied to nonaqueous solvents without ammonium acetate or similar volatile salt, the latter two techniques are essentially identical with DLI operation (see above). Analytical Chemistry, Vol. 66, No. 12, June 15, 1994
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Over the past 10 years the TS interface and its variants have dominated LC/MS applications, and it is now a mature technique. Its principles of operation are well documented even though controversy still exists concerning the dominant mechanism(s) of ion formation. These aspects are admirably covered elsewhere (041-043). It is interesting to note, however, that the principles of flash heating and of prenebulization, discussed above, probably contribute to the success of TS. Arpino (042)has included a thoughtful section on the TS interface viewed as an LC detector. Thermal degradation of analytes in the heated vaporizing tube (generally operated at much higher temperatures than those encountered in the HPN interface) can be a significant problem. The TS interface requires optimization of many operating parameters and, as a result, can be unstable and erratic when operated over appreciable periods of time. The sensitivity is notoriously compound specific, which causes problems in analytical strategies for unknowns. Quantitative analyses are generally considered to require use of a closely eluting internal standard (042).Although the TS interface operates with liquid flow rates up to the 1-2 mL/min range, it is not compatible with involatile buffers. The replacement of ammonium acetate as CI reagent by gaseous ammonia, in the “filament-on” mode, has been advocated for normal-phase LC/MS applications (045). A recent development for reversed-phase LC/MS uses two different capillaries for the volatile buffer and the LC eluate in a dual-beam TS interface, thus separating the thermal requirements of the buffer evaporation and ionization from those of volatilization of the analyte (046). This separation of functions has obvious advantages for gradient elution operation and was found to permit operation with appreciably less thermal degradation. The vast majority of TS interfaces have been designed for quadrupole mass analyzers, but TS interfaces for sector instruments are commercially available though their use is reported rather infrequently. The circular apertures of quadrupole analyzers are well matched to the circular profile of the ion beam emerging from the TS sampling cone. In order to focus this circular ion beam onto the rectangular slit of a sector MS, it is necessary to use appropriate ion optics and a design based on two sets of electrostatic quadrupole lenses has been described (047).The problem of clogging of TS probes, though appreciably less serious than for DLI interfaces, has been addressed (048),resulting in probe designs permitting rapid replacement of blocked capillaries. The introduction in 1981 of fast atom bombardment ionization by Barber et al. (049) revolutionized the applicability of MS to fragile, polar compounds. It has often been commented that the key advance (049) was not the use ofa fast beamof neutral atoms (typically XeO) as the sputtering agent, but rather the application of a viscous liquid (often glycerol) matrix to assist the desorptive ionization. As a result, FAB-MS is often now referred to as liquid-assisted secondary ion mass spectrometry, thus emphasizing the key feature and also accommodating experimental arrangements in which the fast primary beam consists of atomic ions (typically Cs+). In this review the two terms will be used interchangeably. An extensive literature exists, concerning both the mechanisms underlying FAB-MS and the more empirical and more directly useful “tricks of the trade”. It is pertinent to mention here 652R
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a scholarly review of mechanistic studies by Sunner ( 0 5 0 ) and an excellentreview from a more practical viewpoint written by De Pauw et al. (051). Originally FAB-MS was designed as a batch inlet technique, but in 1985 Ito et al. (052)described an LC/MS interface based on the FAB principle. This approach and its further developments have become known as “frit-FAB”, reflecting the original design as a fused-silica capillary incorporating at the MS end a porous stainless steel frit. An extensive study of the effects on sensitivity and stability, of various parameters including balancing glycerol supply rate with evaporation rate, the material, wettability, pore size, and thickness of the frit, has been published (053).A somewhat different approach to application of the FAB ionization principle to flowing liquids was introduced by Caprioli et al. ( 0 5 4 , 055). This approach, which has become known as continuous-flow FAB (CF-FAB), does not employ a frit at the end of the capillary but instead permits the liquid emerging from a capillary to flow over a FAB target. Stability of the MS signal is improved by providing a means (often an absorbent wick) for removal of the accumulating liquid from the target and by controlling the source temperature to prevent evaporative freezing of the liquid. Excellent reviews of the CF-FAB technique have been published by Caprioli et al. (056,057).More recently (058)some CF-FAB probe tip designs were compared; the one found to give the best overall performance appears to combine features of both approaches and has been described in useful detail (059). Ion optics, designed to optimize focusing of ions produced in any FAB source onto the object slit of a sector MS, have been described (060).The features of CF-FAB-MS as a detector for highresolution separatory techniques will be discussed further below, but it is worthwhile to note here its advantages as a batch-modesample introduction system over the original probe inlet design. These advantages include considerable reduction of the “chemical noise” in the spectra arising from the matrix background, considerably less serious suppression of nonsurface-active analytes, and improved mass detection limits derived from injection of the sample as a narrow “plug” into a continuous solvent flow. One of the most striking advances in mass spectrometry over the last few years has been thedevelopment of the matrixassisted laser desorption/ionization technique. The history of the development of this technique has been well described (061),and there is little need to repeat this here except to remark that it seemed unlikely that this approach would provide the basis for an LC/MS interface. However, recently two promising approaches to this end have been described. Li et al. (062)adapted the CF-FAB concept (see above) to MALDI with a time-of-flight instrument, even to the extent that a favored FAB matrix (3-nitrobenzyl alcohol) was employed, replacing the fast atomic beam of FAB by 266-nm light from a Nd:YAG laser operated at 10 Hz. Flow injection experiments were reported (062). The other approach to continuous-flow MALDI is from Murray and Russell (063, LW),whoconstructed aningenious device in which the flowing liquid is pneumatically nebulized, and the solvent removed from the droplets by a heated metal tube which serves to dry the aerosol and to act as a skimmer for the aerosol beam. After transmission into the vacuum chamber of the time-of-
flight mass spectrometer, the beam of aerosol particles, containing both analyte and MALDI matrix, is crossed by IO-Hz pulses at 355 nm of a Nd:YAG laser. Again, results of replicate flow injection experiments were described (063), and a systematic study of the effect on sensitivity of various operating parameters was reported ( 0 6 4 ) . While both ofthese approaches are still some way off from providing a useful LC/MS interface, the results published thus far are promising. Electrospray is the other new ionization technique which has literally revolutionized mass spectrometry over the last few years. This revolution was signaled by the report of Fenn et al. ( 0 6 5 )to the effect that electrospray ionization, on which the authors had worked for several years, could provide mass spectra of intact proteins of molecular mass up to 40 000 Da, bearing as many as 45 charges. The characteristics and scientificantecedents of this breakthrough have been described in excellent reviews, notably those by Fenn et al. ( 0 6 6 , 0 6 7 ) and by Smith et al. (068-070). The essential features of electrospray ionization involve flowing a continuous liquid stream through a metal (typically stainless steel) needle maintained at a high potential (several kilovolts), dispersion of the liquid into charged droplets via electrical instabilities induced in the liquid at the needle tip, and the liberation of ions from the charged droplets, all at atmospheric pressure. An illuminating review by Bruins ( 0 7 1 ) has described the important considerations required in interfacing atmospheric pressure ionization (API) sources (including electrospray and its variants) to mass spectrometer vacuum chambers, and a more recent review by the same author ( 0 7 2 ) provides an excellent perspective on principles and applications of the technique. Early attempts by Eisele ( 0 7 3 ) to improve the sensitivity of API sources by manipulating both gas flows and electric fields to guide a larger fraction of the divergent beam of ions to the sampling orifice were extended by Busman et al. ( 0 7 4 , who also undertook electrostatic calculations. In spite of imaginative attempts, no significant increases in ion signals were observed ( 0 7 3 , 0 7 4 ,a disappointing conclusion ascribed to domination of the electric fields by space charge and by thedominating influence of such fields in theimmediate vicinity of the orifice. However, there has been some success in improving transmission of the ions within the low-pressure region (lo-* Torr or less) between the sampling orifice and the mass spectrometer vacuum. Thus, Douglas and French ( 0 7 5 ) described a means of ion transmission in this region via an rf-only quadrupole which exploits the same collisional focusing phenomenon as is well-known in quadrupole ion trap practice. A scholarly review by Kebarle and Tang ( 0 7 6 ) ,of current understanding of the mechanisms underlying the electrosprayionization phenomenon, has been published.There is no need to summarize these overviews here. Rather, the role of electrospray mass spectrometry as a chromatographic detector will be emphasized in the second cross section of this subject, addressed below. Historically, however, it is interesting to note that the first example of recognizable LC/MS analyses using electrospray ionization appears to have been that published by Aleksandrov et al. ( 0 7 7 ) in 1984, in which a magnetic sector analyzer was used. An independent route into LC/MS using electrospray was provided by the work of Iribarne and Thomson on the physics of the electrical discharge of thunderclouds (078-080), which was later developed into
an “ion evaporation” source for mass spectrometry (081083). This ion evaporation source resembled electrospray in some respects, viz. the flowing liquid was atomized (by a pneumatic sprayer at ground potential in this case) at atmospheric pressure, but differed in the method used to charge the droplets (an electrode at high potential, close to the sprayer tip, induced an opposite charge on the droplets). The potential of this ion evaporation source as an LC detector was investigated in 1983 ( O M ) ,but the sensitivity was too low for trace analysis by LC/MS. This line of development, in the hands of Henion and Bruins ( O M ) ,switched to conventional electrospray as had been proposed for LC/MS applications by Whitehouse et al. ( 0 8 5 ) and culminated in the development of ion spray ionization as a rugged, widely applicable LC/MS interface ( 0 8 6 ) . The ion spray source differs from conventional electrospray in that the atomization of the liquid stream is accomplishedby pneumatic nebulization, rather than relying solely on electrical instabilities. It has been claimed that there is no real difference between electrospray and ion spray, and that the latter name is superfluous. While this is probably a reasonable conclusion for analyses of solutions of purified compounds conducted by continuous infusion into the ion source, it is most certainly not true of LC/MS analyses, the objective of the original development ( 0 8 6 ) . Thus, ion spray permits use of much higher liquid flow rates (up to 200 pL/ min but optimizing at 40-50 pL/min and thus compatible with larger bore LC columns and/or smaller postcolumn split ratios), as well as operation under gradient elution conditions with high percentages of water in the mobile phase. These advantages, resulting from decouplingof the nebulization from the droplet charging, are crucial to the successful application of the electrospray principle to LCjMS coupling. Recently ( 0 8 7 ) the same end was also achieved by using ultrasonic power as the external energy input for nebulization, although as yet this device has been reported to deal only with low flow rates (2 rL/min) of aqueous mobile phase. Lee and Henion (088)have also investigated the effect of directly heating the electrospray needle as an alternative to pneumatic nebulization assistance for electrospray and have further developed the ion spray principle ( 0 8 9 ) to accommodate higher flow rates (up to 2 mL/min) compatible with the full eluate from standard 4.6-mm-i.d. LC columns. Allen and Vestal (090) also incorporated a heated skimmer into an experimental electrospray source, but with the objective of avoiding use of a countercurrent gas flow. Most mass spectrometers incorporating atmospheric pressure ionization sources, including electrospray, have thus far been quadrupole instruments due to their relatively high tolerance for poor vacuum, absence of high potentials, etc. However, implementation of electrospray ionization on sector instruments was reported by McEwen et al. ( 0 9 1 ) . Application of traditional accurate mass measurement techniques to electrospray mass spectra resulted in determinations of molecular mass to within f0.5Da for proteins in the 20-kDa range ( 0 9 1 ) . Cody et al. ( 0 9 2 )have also reported impressive results on accurate mass measurement of individual components of the isotopic peak cluster of a multiprotonated protein. Similar measurements by Starrett and DiDonato ( 0 9 3 ) , on small fragment ions formed in the interface region between the APT source and the vacuum chamber, permitted mass Analytical Chemism, Vol. 66,No. 12, June 15, 1994
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assignments to within 5 ppm. However, none of these measurements (091-093) appears to have been reported as obtained on-line with LC or other separatory technique. Another challenge has been coupling of electrospray sources to FT-ICR instruments (094-098), a major achievement which is not enlarged upon here since it is unlikely to lead to use of FT-ICR instruments as chromatographic detectors in the near future, with the exception of CE (see below). More pertinent in this particular context are efforts devoted to coupling time-of-flight mass spectrometers to electrospray ion sources (099-0103). Some of these designs incorporate ion storage between TOF pulses, thus increasing the duty cycle to close to 100%. The use of TOF instruments with electrospray ionization is potentially beneficial for coupling to capillary electrophoresis,where the time widths of the sample peaks in the electropherogram are sufficiently short that scanning mass spectrometers such as quadrupole mass filters are severely limited in the necessary compromise between scan speed, scan range, and sensitivity. The combination of API ion sources with ion trap mass spectrometers has shown promising results for on-line LC/MS ( 0 1 0 4 , 0 1 0 5 ) . Other notable advances in electrospray practice, in the context of chromatographic detection, include that due to Van Berkel et al. (0106, 0 1 0 7 ) , who successfully produced electrospray mass spectra of polycyclic aromatic compounds and other nonpolar compounds via one-electron oxidation to the radical cations using either electrochemical oxidation or chargetransfer formation with suitable electron acceptors. The latter approach was used ( 0 1 0 8 ) in an on-line LC/MS analysis of a fullerene fraction of condensate from a benzene flame, and for which even HPN-APCI techniques had previously yielded fragment ions of fullerene adducts rather than the desired molecular ions ( 0 1 0 9 ) - The charge-transfer/electrospray ionization technique was sufficiently "soft" that the desired molecular weight information could be obtained, provided that extreme precautions were taken to avoid collisional activation in the interface region. This completes the present survey of recent advances in ionization techniques and peripheral concerns (e.g., sample introduction methods) which have facilitated the use of mass spectrometry as a detector for high-resolution separatory techniques. What follows is an attempt to provide a complementary perspective, organized according to these separatory techniques themselves rather than by the advances in mass spectrometric techniques. This is the second cross section referred to above. SeparationTechniquesWhich Can Use MS Detection. The combination of gas chromatography with mass spectrometry (GC/MS) is a fully mature technique, incorporating E1 and CI as the ionization techniques. Most current activity in GC/ MS development is devoted to designing mass analyzers and associated electronics and computer systems which are sufficiently fast to match the very narrow time-width peaks from the new fast GC technologies (0110-0113). Most of this work is based upon well-known principles established very early in the history of GC (0114, 0 1 1 5 ) ;viz. analysis time can be reduced by 1 order of magnitude through use of very narrow bore capillary columns ( G O hm or so) and shorter column lengths (a few meters). The resulting narrow peak widths implya requirement for fast injection techniques (Dl11, 654R
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0 1 1 2 ) and, of course, fast detectors. A rather different approach to fast GC has been described recently by Rubey ( 0 1 1 3 ) . This technique employs capillary columns of more conventional inner diameter (0.28 mm), thus permitting sample loadings typical of capillary GC, but achieves the rapid analysis times by maintaining a thermal gradient down the length of the column while simultaneously programming the average temperature, thus achieving analyte focusing in a manner somewhat analogous to that in gradient elution HPLC. Both approaches can yield GC peaks only a few tenths of a second wide. Implementation of mass spectrometric detection for such GC separations obviously requires that the spectral acquisition rate be limited to a few milliseconds, and timeof-flight analyzers have been used as the obvious candidates. Enke and his collaborators (0116-01 19) have played a leading role in these developments, exploiting developments due to Wollnik et al. (0120-0122) in efficient ion formation and storage (by EI), and in ion mirror design for mass-independent ion focusing (problems which are much more serious for ionization of gases than for techniques such as MALDI in which the sample is at least at rest in a well-defined spatial location). Very recently ( 0 1 2 3 ) a brief account of a similar approach was published. All of these developments (01160 1 2 3 ) employed E1 sources, as an obvious choice for GC/MS, but Lee et al. ( 0 1 2 4 )have developed an instrument in which an API source was coupled to a time-of-flight MS via a supersonic ion beam arranged to be perpendicular to the direction of acceleration. Application of this system using APCI to capillary supercritical fluid chromatography has been demonstrated (0125). Other developments of GC/MS technology have centered on optimization of on-line postcolumn reactions for selectivedetection of either isotopes such as I3C, 14C, 15N,and 2H (0126-0128), heteroatoms (0129, Dl 30),or halogens ( 0 1 31, Dl 32). The field has been reviewed recently ( 0 1 3 3 ) . This approach is extremely helpful in identifying which peaks in a complex chromatogram are of interest. In the approach developed by Abramson and coworkers (0126-01 32), a microwave-powered chemical reaction interface converts theeluting analytes into small molecules via reaction with a suitable reagent gas; e.g., SO2 converts halogenated compounds to HCl under these conditions. These small moleculesare then selectivelydetected by MS, permitting flagging of the peaks of interest. Finally, an evaluation of a transportable battery-operated GC/MS instrument for onsite environmental analysis has been published (Dl34). Supercritical fluid chromatography (SFC) is often considered to fill a gap between GC and HPLC. A strong driving force to its development in the early 1980s was the belief that interfacing SFC to MS should be simple relative to the problems then facing LC/MS. Much of this early work on SFC/MS was due to Smith and co-workers and to Games et al., bothofwhompublishedreviewson thefieldin 1987 (0135, 0 1 3 6 ) . Two recent articles by Arpino (0137, 0 1 3 8 ) have addressed the question as to why SFC/MS coupling has turned out to be not as straightforward as first hoped. Most early attempts used capillary column SFC with direct introduction of the eluate into essentially conventional CI sources. The low flow rate of mobile phase (almost always C02) could be handled by most conventional MS vacuum systems, and the major problem of providing sufficient heat to the terminating
restrictor tip was readily addressed. Use of the COz as CI reagent gas (charge-transfer ionization) was found to be somewhat restrictive, but postcolumn addition of CI reagent gas was arranged. The major problems of direct capillary SFC/MS include that of increase in gas load on the mass spectrometer near the end of an SFC run using pressure programming, where the analytes of greatest interest for SFC are eluted. Arpino (0137, 138) describes this and other advantages and disadvantages of the technique. An excellent review of this work up to 1989 was published by Sheeley and Reinhold (D139),with examples drawn from this group’s own work on SFC/MS analysis of derivatized oligosaccharides using a magnetic sector MS. Use of direct SFC/MS coupling with packed columns requires the addition of one stage of differential pumping to handle the much greater mobile-phase flow rate, as described by Smith and Udseth (Dl 40) and also by Games et al. (0141) and by Chapman (0142), who used ingeniousadaptations of a thermospray source. An alternative strategy is to remove the SFC mobile phase entirely before introducing the analytes to the MS, and both the moving belt interface (0143, 0 1 4 4 ) and particle-beam interface (0145) have been employed for this purpose. Such decoupling of the optimization of the SFC from that of the MS is advantageous, but the inherent limitations of the moving belt and particlebeam interfaces were found to apply also to their use in SFC/ MS; viz. application to polar and nonvolatile molecules was not possible in general, and sensitivity and quantitation characteristics were poor. The same objective of decoupling theSFC operations from those of the MS can also be achieved, while maintaining direct introduction of the SFC eluate into the ionization source, through use of APCI methodology. The large ventilation rate of the APCI source, which accommodates use of involatile buffers in LC/MS operation as discussed above, also handles the SFC mobile phase as was first demonstrated for packed-column SFC/MS by Huang et al. (0146) and subsequently by Anacleto et al. ( 0 1 4 7 ) . More recently (Dl 48, Dl 49) capillary column SFC/MS using APCI has been demonstrated. It seems likely that the APCI approach will prove extremely useful for future SFC/MS work. Before discussing recent developments in direct coupling of column liquid chromatography with MS, it is appropriate to briefly note the growing sophistication of thin-layer chromatography (TLC) as described in a recent review (Dl 50). Coupling TLC to MS is certainly not new, and indeed at least two MS manufacturers offer an accessory whereby a TLC plate can be scanned using FAB ionization directly from the plate. Reviews of direct TLC/MS coupling have been published recently (0151-0154). The advantages of the additional separatory power afforded by tandem MS for a low-resolution technique such as TLC have been emphasized by Monaghanet al. (0155),althoughdirectTLC/MScoupling was not used. The subject of LC/MS coupling is so large that it is difficult to summarize in a brief space. As emphasized in an illuminating article by van der Greef and Niessen (DI56), modern HPLC techniques encompass the full range of both analyte polarity and molecular mass via normal-phase, reversed-phase, ion-exchange, ion-pairing, and ion chromatographies. This is in marked contrast to GC and SFC,
both of which are applicable to relatively narrow ranges of analyte polarity. Somewhat similar remarks apply to capillary electrophoresis (CE) at the other end of the polarity scale (see below). Fortunately, two excellent monographs describing modern LC/MS techniques have appeared ( 0 4 1 , 0 1 5 7 ) , so the present summary will concentrate on some more recent topics from the literature. The main problem to be addressed in designing an LC/ MS analysis is to ensure compatibility of the LC parameters (mobile-phase flow rate, its polarity and particularly its aqueous content, and modifiers such as buffers, ion-pairing agents, etc.), with those of the MS ion source and/or interface. The moving belt interface, for example, can selectivelyremove mobile phase at the rate of 1-2 mL/min, but is appropriate only for analytes of moderate to good volatility and thus of moderate polarity, which are generally not compatible with highly aqueous mobile phases. An interesting recent application of this principle was the development of on-line LCcombustion isotope ratio MS (DI58), for which reliable removal of mobile phase is essential for high-precision isotope ratio determinations. For this reason a disposable moving wire, rather than a recirculating belt, was used ( 0 1 5 8 ) as the transport mechanism. The thermospray interface is also compatible with large flow rates, but its well-recognized problems (see above) have led to a marked decrease in its use due to the rise in popularity of electrospray and ion spray, as documented (0156) in terms of contributions to ASMS conferences from 1988 to 1991. Over the same period the use of CF-FAB decreased only slightly (0156) at a level about half of that of the combined thermospray/electrospray contributions. The CF-FAB technique is mostly restricted to analytes of moderate to high polarity, normally separated using reversed-phase LC conditions, but its main disadvantage derives from the limited flow rate (about 5 pL/min) which can be accommodated. This implies either a large postcolumn split if conventional HPLC columns are used (1-4.6-mm i.d., with corresponding optimum flow rates in the range 50 pL to 1 mL/min) or else restriction to microbore packed columns with direct coupling. The latter option has both advantages and disadvantages (see introductory comments to this section). Analyte focusing by injection in a solvent of low eluting strength followed by gradient elution can greatly alleviate the limitations imposed by limited sample loading (Dl 59). Like thermospray, CF-FAB is incompatible with involatile mobile-phase modifiers. Also, CF-FAB requires continuous addition of FAB matrix, which can be conveniently achieved by incorporating the matrix in the mobile phase. However, addition of viscous materials such as glycerol can seriously affect chromatographic performance, as detailed in a series of papers by Bertrand and co-workers (Dl 60-01 62). The convenienceof this precolumn addition approach must therefore be weighed against its poorer chromatographic performance, relative to that of the more difficult method whereby the FAB matrix is delivered independently to the probe tip. An efficient method for microcolumn LC involves a coaxial arrangement for delivery of the two independent flows, as described by de Wit et al. (Dl 63) andevaluated relative to precolumn addition of matrix by Pleasance et al. (DI64). An interesting comparison of CF-FAB with ion spray, as a basis for LC/MS analysis of proteins and glycoproteins, has been published by Hemling Analytical Chemistry, Vol. 86, No. 12, June 15, 1994
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et al. (0165). Space does not permit a discussion of this realistic, user-oriented comparison, but it is worth mentioning that the authors concluded that, for this application, ion spray was clearly their method of choice despite a potential greater difficulty in analyzing the data (0165). The CF-FAB method has been widely used for qualitative LC/MS, but very little appears to have been published on its application toquantitative trace analysis. This is in marked contrast to the ion spray method (see below). The two API techniques discussed above, viz. HPN-APCI and electrospray/ion spray, complement one another well with respect to polarity of analytes and thus of the mobile phase. The former can tolerate flow rates in the milliliter per minute range and also involatile modifiers. Electrospray is limited in practice to flow rates similar to those characteristic of CF-FAB and, like the latter, is incompatible with involatile buffers. The additional degree of freedom provided by independent nebulization permits ion spray LC/MS to be operated at 50 pL/min (entireeluatefrom a l-mm-i.d. column) with only moderate loss of ionization efficiency and only minor dependence on mobile-phase composition in gradient elution mode. The question of overall sensitivity of LC/MS analyses using electrospray/ion spray is complicated. It was shown (0166)that an ion spray source has some of the characteristics of a concentration-dependent detector, in that halving the sample flow rate resulted in almost no change in chromatographic peak height (maximum analyte concentration unchanged) but doubled the peak area (see the introduction to this section). No mass spectrometer can be a true concentration-dependent detector like a light absorption (Beer-Lambert law) detector, since halting the sample flow will reduce the MS signal to zero but leave the other unchanged. However, the effect of flow rateon chromatographic peakarea is marked (0166) and probably reflects a decrease in ionization efficiency (coulombs per microgram) with increasing flow rate. This phenomenon has been more completely characterized by Hopfgartner et al. (0167). Interestingly, these workers showed that the high-flow ion spray technique, which they had developed previously (089), behaved as a mass-flowsensitive detector. This work is important (0167) and should preferably be repeated for all LC/MS interfaces with pretensions to applicability to quantitative analyses. It emphasizes the importance of treating the LC/MS system as a whole, for considerations of sensitivity as measured by both theresponsecurves (peakareavs peak height) and by detection limits. Larger flow rates also imply larger inner diameter columns and larger permissible sample loadings, thus impacting directly on concentration detection limits. As emphasized above, the question of LC/MS sensitivity is highly complex and interacts with optimization of chromatographic performance. Some of the compatibility problems can be alleviated by what has come to be known as the “phase-switching” approach of van der Greef et al. (0168),based on heart cutting using valve-switching and coupled-column techniques ( D l 56). As an example of a less common form of LC coupled to MS, the problems of interfacing ion chromatography (0169) with MS have been addressed for thermospray (0170), ion spray (0171), and particle-beam (0172) LC/MS interfaces. In all cases it was necessary to provide for continuous 656R
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postcolumn desalting prior to introduction of the eluate into the LC/MS interface. This was achieved using suitable micromembrane suppressors, which did not appear to contribute significantly to peak broadening. Ion chromatography and ion-pairing chromatography are also frequently used for inorganic speciation in on-line combination with inductively coupled plasma MS or microwave-induced plasma MS. This subject lies somewhat outside the traditional boundaries of the present review, but some recent reviews of the subject are noted here (0173, 0174). An entirely different problem, that of determination of molecular mass distributions of synthetic polymers, has recently been addressed by Prokai and Simonsick (0175) using an on-line combination of gel permeation chromatography (GPC) with electrospray MS. The conventional GPC technique uses a single-parameter detector (e.g., refractive index or UV/visible absorption) to compare the elution profile of the unknown with that of one or more calibration standards. Suitable standards are usually not available, and systematic errors are well-known to occur. Use of an electrospray GPC/MS interface for this purpose required development of an electrospray source which would operate for the organic solvents used in GPC, and the resulting technique provided (M nNa)*+ ions. Use of reconstructed single-ion chromatograms from the GPC/MS analysis permitted a reliable correlation between elution volume and molecular mass to be constructed (0175) for the actual sample under investigation, and this permitted more reliable interpretation of the conventional GPC data. The mass spectral profile could not be used directly in view of known dependences on molecular mass of the ionization efficiencies and of the transmission characteristics of the quadrupole mass filter (0175). Even more unusual was the direct interfacing of high-speed countercurrent chromatography to MS via frit interfaces permitting EI, CI, and FAB ionization (0176). Capillary electrophoresis is rapidly growing in popularity in both chemical and biochemical analysis due to its highresolution separatory capabilities with relatively short analysis times, as well as its amenability to the use of extremely small sample volumes (0177). The separation mechanism involves the interplay between the electroosmotic flow, induced by the applied field acting on the electrical double layer at the liquid/ silica wall interface, and the electrophoretic mobilities of the analytes. Since the latter are nonzero for charged species only, CE is best suited to highly polar compounds so that the obvious choices for interfacing to MS are CF-FAB and electrospray. In fact, electrospray was the first to be used for this purpose by Smith and his collaborators (0178) and independently by Henion et al. (0179). These two groups adopted rather different approaches to the twin problems of accommodating the continuity requirements of the electrical connectionsfor the C E and for the electrospray needle, together with providing a suitable make-up flow to the electrospray source to augment the very low liquid flow rates from the CE column. These “coaxial”and “liquidjunction” interfaces have been described and compared in a review by Pleasance and Thibault (0180). Similar approaches to CE/MS coupling by CF-FAB have also been described (0181-0183). Electrospray has an advantageover CF-FAB for CE/MS coupling because the ion source operates at atmospheric pressure, so that no problems arise with pressure-driven hydrodynamic
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flows distorting the essentially plug flow characteristic of electroosmosis, thus degrading the peak shapes. As for LC/MS, it is important when designing a CE/MS experiment to accommodate the conditions for CE separation and those of the MS ionization. For example, CE separations conducted under conditions with appreciable electroosmotic flow have the ability to elute cationic, neutral, and anionic species in a single electropherogram, and this places heavy demands upon the ionization technique. A more common related problem arises when the CE separation is optimized for, as an example, the anionic forms of the analytes while the best MS response is obtained for the cationic forms. Enhancement of the MS response can then be achieved by the postcolumn addition of appropriate sheath buffers, as exemplified by the work of Moseley et al. (0184),who used CE separation of anionic chemotactic peptides followedby positive ion detection using CF-FAB. Alternatively, the CE separation can be obtained for the cationic forms by derivatizing the silanol groups on the capillary walls as aminopropyl silyl derivatives (0184) or by coating the walls with cationic polymers (0185)to prevent adsorption, thereby facilitating CE separation of these basic compounds at pH 1000 using helium and argon as collision gases. For a given laboratory frame-ofreference collision energy, the overall decompositionefficiency and the specific yield of side-chain fragments (such as d ions) were both greater using argon, consistent with vibrational rather than electronic excitation as the principal activation mechanism (E16). Downard and Biemann (E1 7) have studied the formation of C-type ions from protonated peptides. Such ions are commonly observed in conventional FAB-LSIMS spectra but less frequently in CAD product ion spectra. A study of highenergy CAD of protonated peptides revealed a propensity to C-type cleavage preceding threonine, and to a lesser extent tryptophan, lysine, and serine, when N-terminal fragmentation is favored by other structural features. A limited stable isotope labeling study suggested that the mechanism of formation of c ions involved hydrogen transfer from carbon; the proposed mechanism incorporated hydrogen transfer from the first carbon of the threonine (or other residue) side chain ( E I 7 ) . A similar mechanism was proposed by van Dongen et al. (E18), also based on deuterium-labeling studies. A previously unrecognized fragmentation of protonated peptides was reported by Thornburg et al. (E19). High-energy collisional activation in the first or third field-free regions of a foursector instrument generated (a2 - 16) ions; linked scanning of first field-free region decompositions demonstrated formation from (a2 + l ) and (b2 - 16) precursors, presumably by loss of NH3 and CO, respectively. Several factors have encouraged a continued high level of research into the decomposition of peptide ions under condi058R
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tions of low-energy CAD. Most commonly, the experiments are performed using triple quadrupole or hybrid instruments which are significantly less expensive than four-sector instruments. Despite the inability to detect multiple ion species simultaneously (in contrast, for example, to the use of array detectors on four-sector instruments (E20)),effective tandem MS analyses of peptide mixtures have been performed with very high sensitivities (tens of femtomole level) using a triple quadrupole instrument, albeit with a substantial sacrifice in precursor and product ion resolutions ( E 2 1 4 2 5 ) . Electrospray ionization is most commonly (though by no means exclusively) installed on quadrupole mass spectrometers. Finally, the increasing interest in tandem MS using the ion trap mass spectrometer ( E 2 6 4 3 1 ) has further emphasized the need for improved understanding of low-energy decomposition processes (though the time scale of the ion trap experiment may be extended substantially beyond the range typically involved with decompositions in a linear quadrupole (or other multipole)). There is now explicit recognition that the extent of decomposition observed in tandem MS/low-energy CAD experiments is influenced by the internal energy of the precursor ions acquired during the ionization process. Thus, for example, Kilby and Sheil (E32) showed that the decomposition efficiency of electrosprayed ions following low-energy CAD in a triple quadrupole was increased when a relatively high skimmer potential was used in the interface region. Collisional activation in the electrospray interface has been quite widely used to promote prompt decompositions in that region ( E 3 3 4 3 7 ) ;the Kilby and Sheil study (E32) demonstrated that a significant proportion of ions are activated but remain intact, at least during passage through the first quadrupole of the tandem instrument. McCormack et al. (Ed) demonstrated a greater extent of decomposition of precursor ions followingsurface collisionsin a triplequadrupole when LSIMS was used rather than electrospray ionization. In general, the fragmentations of peptides observed under low-energy CAD conditions are qualitatively similar to those which occur spontaneously. Thus, low-energy CAD may increase the yield of fragment ions but does not usually open new fragmentation pathways. The followingdiscussion relates to low-energy processes, whether or not promoted by collisional activation of the precursor. (It is noteworthy that low-energy processes may make a substantial contribution to the product ion spectrum recorded under conditions of high-energy collisional activation (E38).) In an early landmark paper describing FAB-tandem MS of peptides using a triple quadrupole instrument, Hunt and co-workers (E39) briefly proposed a model for low-energy decomposition processes encapsulating key issues which have been emphasized in more recent studies. Charge delocalization in protonated peptides was envisaged by Hunt et al. (E39) to occur by “internal solvation”, corresponding to folding of the peptide chain to bring into proximity apparently remote portions of the structure. It was proposed that the energy barrier to transfer of a proton from the N-terminal amino group to an amide bond was quite small, facilitating the generation of a variety of structures to promote amide bond cleavage. It was additionally suggested that a lower internal energy of the precursor favored cleavage of the amide bond to yield a y-type
ion (E40, E41), in preference to a b type, since formation of the former involved both bond cleavage and formation; redistribution of the proton within the product ions was, however, anticipated to result from equilibration within the long-lived product ion/neutral complex. (Deuterium labeling has been used to probe the precise mechanism of the formation of y-type ions; a hydrogen atom attached to a nitrogen, rather than to a carbon, is transferred during cleavage of the amide bond (E42, E O ) . ) Typical conditions of low-energy CAD promote multiple collisions which, in combination with the extended time scale, favor the occurrence of multistep cleavages. “Internal” fragment ions, for example, are quite commonly observed, resulting from sequential cleavageof two amide bonds (E39, E44). Recent work has substantiated the hypothesis that lowenergy fragmentations are principally charge-directed, The site of charge may be effectively fixed, either by incorporation of a highly basic site such as the arginine residue or by the formation of a precharged derivative ( E 4 5 4 4 8 ) . Burlet et al. (E49) demonstrated that the fixing of charge site was not conducive to the observation of diagnostic low-energy fragmentations. Favorable fragmentation properties could be restored by the formation of doubly charged ions via further protonation. (See the discussion below of the fragmentations of doubly charged ions.) The situation is distinct from that which applies to high-energy decompositions of peptides incorporating a precharged derivative group. Work in several laboratories has shown that high-energy CAD of such derivatives can provide simplified and structurally informative product ion spectra, presumably by favoring charge-remote processes (E45, E50,E51). Explicit evidence for the influence of gas-phase conformation of peptide ions on low-energy decomposition processes has come from recent work. Burlet et al. (E52),for example, attributed marked differences in the low-energy decompositions of cysteine- and cysteic acid-containing peptides incorporating a C-terminal arginine to an interaction between the cysteic acid side chain and the strongly basic arginine side chain. Such interaction effectively attenuates the basicity of the arginine residue and allows distribution of the proton between alternative sites (principally amide bonds), facilitating multiple sites of cleavage. The favored production of y-type ions may result from the lower energy requirement for such cleavages or may reflect a shifting of the charge to the more basic fragment during the lifetime of the charged/neutral complex (E39). Intraionic interactions are also indicated by the observation of oxygen isotope exchange between the C-terminal carboxyl group and the first amide bond of the protonated peptide (E53). Intraionic 180-exchangehas also been observed between the C-terminal acylium ion (resulting from C-terminal loss of H2180 from the carboxyl-labeled peptide) and various locations on the peptide chain (E54). Bursey and co-workers have invoked thermochemical arguments to rationalize the low-energy fragmentations of simple protonated peptides ( E 5 5 4 5 7 ) . These authors argued that, in peptides lacking proline and amino acid residues with basic side chains, the intrinsically most basic site is the N-terminal amino group, with basicity enhanced by the formation of hydrogen bonds (E55).Protonation of an amide bond (to yield C(OH+)NH) may, however, become compa-
rable in energetic terms as a result of internal solvation. Certainly, the prevalence of b-type ions in longer chain ( 5 - , 6-, and 8-mer) homopolymers of alanine suggests amide bond protonation (E55,E56). In proline-containing peptides such as Ala2ProAla2, thermochemical arguments indicate favored formation of the y3 ion, with protonation on the proline N (E56). Such arguments are consistent with the common observation of preferential cleavage N-terminal to proline residues (E39). Independent evidence for the significance of hydrogen bonding in determining the gas-phase properties of peptide ions derives from the workof Cheng et al. (E58)on the collision energy dependence of the dissociation of proton-bound dimers incorporating Gly3, Glyd, and various bases (B). Determination of the relationship between the relative abundances of the protonated monomers and the center-of-mass collision energy (which determined the effective temperature of the proton-bound dimers) allowed estimates of the differences in proton affinities and in the differences in the entropy of protonation. Dissociationof [B + Gly3]H+ and of [B Gly41H+ showed collision energy dependence indicative of pronounced entropy effects which were attributed to intraionic hydrogen bonding. The use of this kinetic method (involving the observation of decomposition of protonated heterodimers (E59))for the rigorous determination of gas-phase basicities and proton affinities remains controversial. The approach has undoubtedly been useful, however, in generating comparative data which can contribute to the understanding of peptide fragmentation. Thus, for example, Wu and Fenselau (E6O)compared thegas-phase basicities of a series of peptides incorporating a basic residue and found a minor effect of basic residue position on peptide basicity in the order amino terminal > internal > C-terminal. This finding was interpreted to indicate the different degrees of charge delocalization resulting from multiple interactions with the proton (E60). Wysocki and co-workers ( E 5 )have described a theoretical approach to the explanation of fragmentations of protonated peptides, based on MNDO bond order calculations. Protonation was demonstrated to have only a local effect; bond orders a,8, and y to the site of protonation were affected. Protonation of the amide nitrogen weakened the amide bond whereas protonation of the amide oxygen (predicted on thermochemical grounds (E55)to be the more basic site) strengthened the amide bond. These calculations suggest that low-energy, charge-directed fragmentations of protonated peptides are promoted by multiple sites of protonation, consistent with the experimental data summarized above. The data also suggest the need to consider structures of the protonated peptides other than those predicted thermochemically to be the most favorable. The same study indicated that charge-remote fragmentation (modeled by consideration of the neutral peptide) was energetically less favorable than amide bond cleavage local to the site of protonation; again the finding is consistent with the relatively high energy requirement for d and w fragment ion formation (E61). MNDO diatomic energy contributions were subsequently demonstrated to provide estimates of bond strengths equivalent to the bond order calculations (E62). A problem in reconciling theoretical predictions with experimental evidence has been the difficulty in defining the
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energetics of decomposition experiments in tandem MS.SID, however, has been demonstrated to result in a much narrower range of energy deposition than is typical of CAD (E2, E63). Cole et al. ( E 3 ) recently demonstrated that the range of fragmentation processes observed in an FAB-SID experiment was attributable to the translational energy spread (estimated at 7eV) of the desorbed ions which served as precursors in the tandem MS experiments, rather than to a variation in energy deposition accompanying surface collisions. Collisional cooling of ions in the interface region of an electrospray ionization source is expected to provide a population of relatively cold precursor ions with a narrow range of kinetic energies. Jones et al. (E64) have recently demonstrated a lesser decomposition efficiency of LSIMS-desorbed than electrosprayed protonated peptides. Thus, the combination of electrospray and SID provides the potential for more precise definition of the energetics of the tandem MS experiment. In the study by Jones et al. (E64),an investigation was made of the surface collision energy onsets of fragmentation of various electrosprayed protonated peptides differing in amino acid sequence. The data showed that the absenceof a basic residue resulted in a populationof protonated peptides that fragmented more easily at a given collision energy. Among argininecontaining peptides, a structure incorporating a C-terminal arginine fragmented more readily than a peptide with the arginine at the N-terminus. The experimental data were rationalized in terms of the energy requirements for intraionic proton transfer from the most basic residues to alternative sites (such as amide bonds) where fragmentation is promoted. When arginineis incorporated at theN-terminus, the preferred protonated structure is envisaged as proton-bridged between the basic side chain and the N-terminal amino group, with consequent enhancement in the energy requirement for proton transfer (E64). The general concept of the requirement for proton “mobility” to promote multiple diagnostic cleavages is in keeping with earlier experimental data and nicely complements the modeling studies discussed above. Thus, a number of recent studies have illustrated the complementary value of thermochemical, molecular modeling, and experimental approaches to the study of peptide fragmentation. Thermochemical arguments (taking due note of intraionic interactions) can suggest the proportions of the precursor ion population incorporating protonation at different sites and can indicate energetically favored fragmentation pathways. Molecular modeling addresses the propensities to fragmentation of the different forms. The interpretation of the experimental data must clearly acknowledge the effects of both precursor ion population and fragmentation tendencies. The ideas that have emerged from experimental and theoretical studies of singly protonated peptides provide an appropriate starting point for the explanation of the decompositions of multiply charged peptides, the study of which has become important following the development of electrospray ionization. Thus, for example, peptides derived from tryptic digestion of proteins (and therefore incorporating a basic amino acid residue at the C-terminus) have been generally observed ( E 6 5 4 6 7 ) to yield prominent doubly charged ions by electrospray, consistent with condensed-phase protonation of the basic side chain and of the amino group at the N-terminus (E68). An early suggestion (E67) was that this charge 660R
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distribution was retained in the gas-phase ion, implying that complementary series of singly charged band y ions, observed following low-energy collisional activation, resulted from charge-remote cleavage at various points in the peptide chain. This mechanism appears not to be consistent with the experimental and theoretical studies of singly protonated species discussed above. As Boyd and co-workers (E69)have pointed out, however, initial protonation on a basic side chain and at the N-terminus does not necessarily imply that this represents the charge distribution in an activated gas-phase ion. Thus, whileone protonmay besequestered by thestrongly basic amino acid side chain at the C-terminus, the second proton may occupy one of many possible sites. This suggestion is entirely consistent with the ideas derived from study of the low-energy decompositions of singly protonated peptides, namely, that a ”mobile” proton may promote a variety of fragmentations (E49, E64). The observation of b and y series of singly charged ions from doubly protonated tryptic peptides (E66)has prompted the question as to why these apparently complementary ions are generally observed with relative abundances strongly favoring they series. Tang and Boyd (E68)presented evidence to eliminate [M 2H]’+ and [M + H]+ as intermediates in the formation of y ions and additionally established (by CAD of the y- and b-series fragment ions formed in the electrospray interface region) that the y series showed a much lesser tendency to further fragmentation than the complementary b ions. Again, this finding was consistent with observations with singly protonated peptides since y ions derived from decomposition of tryptic peptide [M 2HI2+ions are formally equivalent to protonated peptides with an expected site of protonation on the basic side chain of the C-terminal residue. Tang et al. (E69) have also described a detailed study (using a triple quadrupole mass spectrometer) of the decompositions of a number of peptides in several charge states, derived by multiple protonation. In multiply protonated peptides with several basic residues, the extent of decomposition was low, consistent with the sequestering of protons by the basic side chains. In the absence of a strongly favored location for one or more protons, the observed charge-initiated fragmentations suggested an influence of Coulombic repulsion in determining charge site on the peptide backbone. Coulombic repulsion was not itself, however, the driving force for fragmentation. The same authors (E69)reported a fascinating rearrangement process involving the transfer of one or two amino acid residues from the C-terminus of [b HI2+fragments to the side chain of a lysine residue located near the N-terminus, resulting in mass shifts of the subsequent second-stage fragmentations. There have been few reports of the high-energy CAD of multiply charged peptide ions, reflecting the greater practical difficulty of installing electrospray interfaces on sector instruments. Orlando and Boyd (E70) have discussed the requisite laws for scanning of product ions of differing charge states formed in a floated collision cell from a multiply charged precursor. The data presently available for high-energy CAD of multiply protonated peptides (E71)indicate that side-chain cleavages (d and w ions) are observed in addition to peptide backbone fragmentation, paralleling observations for the singly charged counterparts. As noted above, the contribution of low-energy processes to the total product ion spectrum recorded
+
+
+
under high-energy CAD conditions is not, in general, known. In a series of papers (E72-E75), Smith and co-workers have used a triple quadrupole instrument to examine the CAD of multiply charged ions with masses as high as 66 kDa. Fragmentation of ions this large represents a remarkable achievement but the data present formidable problems of interpretation (even for a known sequence) as the mass and charge state of each product ion are not, in general, independently determined. Several useful, but inconclusive, strategies to address these problems have been described (E75); they include the search for complementary fragments, the CAD of fragment ions formed in the electrospray interface, and the comparison of data from structurally related analytes. Although the sequence information evident from the product ion spectra of multiply protonated albumins (M,66 000) was limited, the data permitted empirical distinctions between albumins derived from different sources. In studies of several multiply protonated proteins, relatively facile cleavage Nterminal to a proline residue was observed (E74, E75), reproducing observations (as discussed above) for the fragmentations of singly protonated small peptides. Presumably the explanation is also the same, namely, the enhanced basicity of the proline amide bond, facilitating charge-proximal cleavage. In summary, the study of the decompositions of peptide ions remains an area of continuing activity. Theoretical and experimental approaches have recently shown an encouraging convergence, but the full explanation of the fragmentations of multiply charged ions represents a substantial challenge.
F. PEPTIDES AND PROTEINS The structural analysis of peptides and proteins usually requires the use of a multifaceted strategy involving separate steps for purification and cleanup, selective modification and degradation, separation, and sequence and structural characterization (Fl , F2). Achieving and maintaining solubility of all components and minimizing losses throughout these manipulations can be a considerable challenge, such that monitoring independently the effectiveness of individual steps is prudent or even necessary. The exact approach will vary with each new protein because of the incredible diversity of their chemical properties and behavior. These sample handling problems are only exacerbated when working at the 10-50 pmol level, despite the fact that certain mass spectrometric techniques have absolute detectibilities some 3 or 4 orders of magnitude lower. Mass spectrometry now has a role to play at every level in solving these problems in protein research, from the assessment of global purity and detection of heterogeneity in the original sample to the eventual careful, specific accounting for every single mass unit (F3). This point is nicely illustrated by work on the characterization of a recombinant phosphorylated variant of the thrombin inhibitor, hirudin, using electrospray ionization in a variety of modes (F4). It is helpful in defining the precise context within which all other data must fit. At this juncture, tackling these problems without the involvement of mass spectrometry is certainly a disadvantage and, when facing issues of the nature and extent of covalent modification, could be a time-consuming liability. While the advantages and growing importance of such mass spectrometric-based
strategies are not extensively appreciated currently in the graduate school education in most universities, fortunately there will soon be authoritative introductory volumes which include these topics (F2). To be sure, there is some balance between those aspects of cell biology which are in the purview of the molecular biologists(the human genome at oneextreme) and those which are of necessity the realm of protein biochemists, e.g., activation, translocation, regulation or selective inhibition of proteins by covalent transformations of their structures, etc. Recognition is growing that mass spectrometry can fill the void in detecting and delineating covalent modulation, a topic these established disciplines were not able to address in a succinct, authoritative manner previously (F5). There is certainly room for movement toward symbiotic integration of efforts in molecular biology and the techniques of protein biochemistry, mass spectrometry, and biotechnology to effect more rapid realization of overall scientific goals, especially in the context of the discovery of protein function and rational drug design. Such movement has become apparent from the astoundingly rapid increase in the number and quality of reports involving the use of mass spectrometry at annual meetings of The Protein Society during this review period (Fb,F7). These meetings provide an excellent forum for crossfertilization of these disciplines and honing an interplay between laboratories traditionally focused on either mass spectrometry or sequencing and cloning, whether they have academic or biotechnological interests. In this regard the Third International Symposium on Mass Spectrometry in the Health and Life Sciences will take place September 13-18, 1994, in San Francisco and will focus again on this macromolecular theme (F8). During this period, a number of treatments (F2, F3) and reviews (F9-F20) have appeared which emphasize different aspects of techniques and achievements and have struck somewhat different themes on how mass spectrometry and protein biology go together. Amino Acids. The derivatization of amino acids was one of the very early forays to adapt labile zwitterionic substances through direct vaporization into an electron impact ion source for mass spectral characterization (F21). Recently, a onestep procedure based on an aqueous-phase chloroformatemediated derivatization has been reported for complete amino acid analysis by both GC-FID and GC/MS (F22,F23).Work on improving sensitivity of the Edman degradation using a quaternary ammonium phenyl isothiocyanate and electrospray detection has been reported (F24). E-N-trimethyllysine appears to be present in storage body protein of sheep with juvenile ceroid-lipofuscinosis (Batten disease) (F25). Synthetic Peptides and Proteins. As one would expect, work continues to improve the effectiveness of peptide resin cleavage and deprotection following solid-phase synthesis (F26).Theuseof mass spectrometry is essential in establishing the authenticity of such synthetic material. Measurement of molecular weight and purity ( F 2 7 4 3 0 )should be followed up in those cases that are appropriate by high-energy CID analysis (F3I). In particular cases, low-energy CID data may be adequate (F32). Higher molecular weight synthetic products may be analyzed by electrospray (F33, F34) or matrix-assisted laser desorption (F35). However, one should Analytical Chemistry, Vol. 66,No. 12, June 15, 1994
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be cognizant of the report that oxidation of methionine can take place during electrospray ionization (F36). Bioactive Natural Peptides. In continuing studies of microcystis obtained from cyanobacteria, Rinehart and colleagues have reported a series of new structures from a lake bloom during the midwestern United States drought in 1988, which are hepatotoxins and protein phosphatase inhibitors, making them important biological tools (F37). Studies of N-terminally blocked neuropeptidesin the earliest animal group having a nervous system, thecoelenterates, have revealed that the biosynthetic machinery is already very efficient and analogous to that of higher invertebrates (F38-F40). These neuropeptides, as well as other neuropeptides isolated from corpora cardiaca of various cetonic beetle species (F4I),have been sequenced by tandem mass spectrometry. Further work on the potent antitumor ecteinascidins reveals DNA stacking interactions similar to other minor groove binders (F42). Tandem mass spectrometry has also been applied to structure determination of macrolide antibiotics (F43). Mass spectrometry has played a role in characterizing a variety of other peptide natural products, including a neuropeptide Y homologue that produces prolonged inhibition in Aplysia neurons (F44);cycloamanide peptides with immunosuppressive activity from mushrooms (F45);a tridecapeptide amide bacteriocide from neutrophils (F46);a calcium channel blocking, spider toxin, o-agatoxin IA (F47); a sperm-activating peptide type-V from the heart urchin, Brissus agassizii (F48);a vitellogenesis-inhibiting hormone from the Mexican crayfish, Procambarus bouvieri (0rtmann)(F49); a pigment-dispersing hormone of the shore crab, Carcinus maenas (F50);diuretic hormones from Locusta migratoria ( H I ) and a deoxyhexose-linked peptide from the same species (F52,F53);and a succinyl tryptophenylsubtilin antibiotic (F54);and a large number of peptides from other cyanobacteria (blue-green algae) (F55462).In addition, a new group of potent cytotoxic and antiviral compounds, the crambescidins, have been described, consisting of a pentacyclic guanidine moiety linked by a long-chain o-hydroxy acid to a hydroxyspermidine or spermidine unit (F63). PosttranslationalModifications. There have been further reports on a variety of posttranslational modifications. The major component of bovine liver acyl-CoA-binding protein was shown to be N-terminally acetylated, while two minor variants were shown to be acetylated in one case and malonylated in the other case at Lys-18 (F64). Stefin B and two low molecular weight phosphotyrosine protein phosphatases from rat liver are N-terminallyacetylated(F65, F66). A semiautomatedchromatographicprocedure for the isolation of acetylatedN-terminal protein fragments has been described (F67). Human interleukin-5 was shown to have a variety of modifications, including N-terminal formylation and carbamoylation (F68). An insulin-relatedpeptide from Lymnaea stagnalis (F69) and fibrolase from Agkistrodon contortrix contortrix (F70) have N-termini blocked with pyroglutamic acid. The DNA-binding protein Ner of bacteriophage Mu is blocked with a pyruvic acid moiety (F7I). Electrospray mass spectrometry revealed the fact that the amino terminus of retinal recoverin is acylated by a family of fatty acids (F72). In addition, rod transducin CT subunit 662R
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is acylated by a heterogeneous group of fatty acids (F73),and the N-terminus of human myelin basic protein is modified by fatty acids from CZto C ~ (F74). O Finally, the CT subunit of the photoreceptor G-protein transducin, Ta, is acylated with a series of both saturated and unsaturated fatty acids, C l r C14 (F75). Differences in the primary structure of human surfactant-associated proteins isolated from normal and proteinosis lung have been shown to be due to partial or complete removal of cysteinyl-5,6-thioester palmitate residues present in normal lungs and further N-terminal proteolytic degradation (F76). The outer surface protein A, OspA, is an interesting lipoprotein antigen from the spirocheteBorrelia burgdorferi, the causeof tick-borne Lyme disease. The N-terminal cysteine residue is palmitoylated, and the side-chain thiol is alkylated by a 2,3-bis(palmitoyloxypropyl)moiety (Pam3Cys). The molecular weight of theintact recombinant OspA was obtained using electrospray mass spectrometry after removal of detergent by acetone precipitation and subsequent solubilization in pure formic acid. The solution was then diluted with a mixture of 2-propanol/methanol/waterfor introduction into the electrospray ion source. The major component observed appears to be the fully lipidated product, with the presence of 30% of a component lacking one palmitic acid residue and a small amount of protein with an unmodified N-terminal cysteine residue (F77). A further important paper reporting methodology permitting the analysis of hydrophobic peptides and protein by electrospraymass spectrometry has recently appeared (F78). Identification of the geranylated y subunits of guanine nucleotide-binding regulatory proteins has been carried out on the modified C-terminal pentapeptide by FAB-MS (F79). High-energy CID analysis was used to establish the posttranslational modification of the mating factor nonapeptide from fission yeast, Schizosaccharomycespombe. In this case, the C-terminal cysteine residue is modified by farnesylation and conversion to the methyl ester (F80).Twodifferent forms of monomeric NADP+-linked prostaglandin dehydrogenase/ carbonyl reductase were purified from human placenta. Mass spectrometry was used to detect the modified residue, which wassubsequentlyestablished to be N6-(1arboxyethy1)lysine at position 238 in one form of the enzyme (F81). FAB-MS was employed to establish the polyglutamation of glutamic acid 435 in the major brain @ tubulin isotype class I1 tubulin (F82). As is well-known from the literature, asparagine-glycine sequences are prone to deamidation. Two different sites and two isoforms of recombinant hirudin have been characterized by electrospray mass spectrometry (F83) and by collisioninduced dissociation of '*O-labeled hydrolysis of the succinimide dehydration products (F84). Using a combination of Edman degradation and plasma desorption mass spectrometry, the heterogeneity in bovine liver fatty acid-bindingprotein was shown to be due tooxidation of cysteine-69 in one case and glutathionylation of the same residue in another case. In addition, amino acid substitution at asparagine-105 for aspartate was revealed through mass measurement of the number of carboxyl functions in the peptides in question by making the methyl ester derivative (F85). Plasma desorption mass spectrometry was employed
to identify the zinc-binding domain in human interleukin-3 (F86). Isoforms of the nuclease from Serratia marcescens was shown to result from truncation of one to three amino acids from the N-terminus (F87). The nature of the amino acid in an insertion mutant of Staphylococcal nuclease was established by MALDI mass spectrometry (F88). MALDI mass spectrometry was also employed in the characterization of the malonyl-CoA-sensitive carnitine palmitoyltransferase of rat heart mitochondrial particle (F89).Mass spectrometry was involved in establishing that molecular oxygen is the source of the oxygen atom in the cy-hydroxyglycine intermediate of the peptidylglycine cy-amidating reaction (F90). Disulfide Linkages. The use of selective derivatization of free thiols with p-hydroxymercuribenzoate and subsequent analysis by MALDI mass spectrometry has been recently reported (F9Z). A variety of techniques for assignment of both intra- and inter-disulfide linkages are in practice based on batch introduction of protein digest fractions and, in more complicated cases, using LC-electrospray to locate particularly large disulfide-linked components. Recent examples include studies of a pentameric structure containing a covalently linked disulfide dimer of rat C-reactive protein (F92),human tissue inhibitor of metalloproteinases (F93),human J chain in secretory immunoglobulin A (F94),human soluble CD23 (F95),the N-terminal domain of the cellular receptor for human urokinase-type plasminogen activator (F96),dimers of bovine cysz-casein(F97),and the complete intra- and interdisulfide bonding in dopamine &hydroxylase (F98). A combination of amino acid composition analysis,sequence determination, and mass spectrometric molecular weight measurement was employed to assign the disulfide linkages of the pheromones Er-1 and Er-2 from the ciliated protozoan Euplotes raikoui (F99). Other proteins which have been characterized include boar spermadhesin AQN- 1 (FI 00)and recombinant human macrophage colony-stimulating factor (FZOI-FZ04). Both electrospray and fast atom bombardment mass spectrometry were used in two of these studies to detect and characterize intermediates involved in the folding pathway of recombinant human macrophage colony stimulating factor (FIOZ,F104). Since this heterodimeric protein has 18cysteine residues which form three intermolecular and two sets of three intramolecular disulfide bonds, the resistance of the native material to proteolytic cleavage was overcome by initial chemical cleavage using cyanogen bromide (FZ03).Subsequent digestion with endoprotease A s p N was then effective in opening the complex structure. Disulfide bond assignments have been reported for baculovirus-expressed mouse interleukin-3 (FZ05) and human and murine interleukin- 10 (FZO6).Using human transforming growth factor-cy as an example, high-energy collision-induced dissociation analybis was shown to be very effective in sequencing through the cystine bridge of intermolecularly disulfide bonded peptides in complex enzymatic and chemical digest of the native protein (F107). Glycosylation. Further work on putative glycosylation site occupancy follows original observations by Carr and Roberts (FZ08).In studies of posttransational processing of membrane-associated recombinant human stem cell factor (FZ09),data have been presented illustrating the use of
incorporation of l 8 0 into the aspartic acid residue formed during cleavage of the N-linked oligosaccharides from asparagine sites (F110).Observation of mass doublets quickly reveals deglycosylated peptides in such a digest, and the 1 8 0 label may be of use in interpreting some collision-induced dissociation processes. One of the more satisfying developments which is already in routine use is the highly successful technique of LCelectrospray analysis of protein digests and, in particular, use of collision-induced fragmentation in the selected ion monitoring mode for the detection of even minor glycopeptide fractions through the observation of specific sugar oxonium fragments (FZZ I ) . This so-called "collisional excitation scanning" coupled with the advantage of use of a different mobile phase in which glycoforms are chromatographically resolved (FI12) represents the most powerful methodology developed thus far for the characterization of complex glycoproteins. Collision-induced dissociation of glycopeptides adds yet another dimension to the power of the electrosprayLC/MS method (F1Z3,F114). Electrospray analysis of glycopeptides has the distinct advantage of mass resolution of glycoforms, even for rather high molecular weight glycopeptides, which would clearly not be mass resolved by MALDI mass spectrometry and probably not even detectable by liquid SIMS (FIZZ,FZZ2,FZZ5). Recently, it has been reported that there is considerable carbohydrate sequence-related information in the high internal energy ions naturally generated in the MALDI process, which can be detected and mass measured by stepping through reflectron energy-focusing potentials in a time-of-flight instrument having such technical capability (FZ16). While it appears that nonreducing terminal sialylation is cleaved in these inherently high-energy MALDI-desorbed molecular ions, the asialoglycopeptides display cleavages of their glycosidic linkages in these reflectron-generated spectra. Further detail on protein glycosylation, in particular glycopeptides, may be obtained from a combination of these mass analysis capabilities and selective enzymatic digestions carried out in a sequential fashion (FZZ2). It is likely that glycopeptide structural analysis will be tractable in the low-picomole and, possibly, femtomole range by these developments using the reflectron MALDI time-of-flight strategy and the new LC-electrospray magnetic sector instruments with multichannel array detection systems. MALDI mass spectrometry has been used to confirm the composition and elution order of five glycosylation site glycopeptides of human cyl-acid glycoprotein (FZ17). Characterization of the N-linked carbohydrate class's glycosylation on normal and congenital dyserythropoietic anaemia type I1 (HEMPAS) transferrin has established that the biosynthesis is altered in HEMPAS hepatocytes from a complex type to a combination of high-mannose and hybrid-type oligosaccharides (FZ18). The presence of a predominantly disialylated biantennary structure on rat C-reactive protein was established by electrospray measurement of the molecular ion distribution and characterization of the permethylated oligosaccharides (FZ19). Characterization of the glycosylation on the nonspecific cross-reacting antigen 160 from CD15positive neutrophil membranes and its identity as biliary glycoprotein 1 has been reported (FZ20). Further work on abnormal glycosylation of serum transferrin has been reported Analytical Chemlstty, Vol. 66, No. 12, June 15, 1994
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in studies of the carbohydrate-deficient glycoproteinsyndrome (F121). MALDI and electrospray techniques have been employed to obtain a glycoform profile of an intact monoglycosylated glycoprotein. These data have been evaluated for batch quality control purposes (F122). The proportions of glycoformswere compared with data from HPLC and proton NMR on the isolated glycans themselves and found to be in good agreement, permitting the assignment of antennicity and degree of sialylation. Several papers have focused on the identificationof O-linked N-acetylglucosamine, including work on vertebrate lens a-crystallins (F123). serum response transcription factor ( F I N ) , and mammalian neurofilaments L and M (F125). In one laboratory, derivatization by propionylation and FABMS has been employed (F123, F124), while in another laboratory, MALDI mass spectrometry has been used to characterize the free glycopeptides directly (F125). Characterization of the O-glycosylation on threonine-445 of the a chain of recombinant human hepatocyte growth factor was determined by a combination of electrospray mass spectrometry and digestionwith neuraminidase (FI26). Further studies of human factor IX has revealed an O-fucose glycosidically linked tetrasaccharide to serine-61 (F127). Using a variety of techniques including plasma desorption mass spectrometry, the presence of a hexasaccharide O-glycosidically linked to tyrosine in the surface layer glycoprotein of Clostridium thermohydrosulfuricum S102-70 was reported (F128). Furt her work using electrospray and high-energy collision-induced dissociation has established the presence of sialic acid on the glycosylinositol phospholipid anchor of scrapie and cellular prion proteins (F129). Phosphorylation. Interest in development and use of mass spectrometric approaches for the detection and sequence identification of protein phosphorylationhas continued. While the advantages of liquid chromatography and electrospray have received most of the attention thus far, matrix-assisted laser desorption mass spectrometry has been employed to identify phosphorylationsites of Opl8 in Jurkat T cells induced by treatment with phorbol 12-myristate 13-acetate (F130). The work was carried out by resolution of the major phosphorylated forms of the protein before and after treatment with PMA using 2-D gel electrophoresis, followed by tryptic digestion and separation of the phosphopeptides. Further subdigestion was carried out when required, and the samples were separated by reversed-phaseliquid chromatography prior to MALDI analysis. Work has been reported on detection of phosphorylated peptides in unseparated digests of human #?-casein (F131). An interesting contribution concerns the use of immobilized ferric ions for selective chelation of several phosphotyrosine and phosphoserine peptides from bovine @-caseinpurified by SDS-PAGE gel electrophoresis and blotted onto PVDF membranes for in situ digestion with trypsin. The entire digest, including buffers, is then analyzed on-line with electrospray mass spectrometry (FI32). In a fashion analogous to work on the detection of the glycopeptides in LC-electrospray/MS runs of glycoproteins, Carr and co-workers have reported similar selected detection of phosphopeptidesin phosphoprotein digests by selected ion monitoring of ions at masses 63 and 79 (F133). This is a very effective strategy for locating and, 664R
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through subsequent use of collision-induced dissociation analysis, identifying the phosphorylation sites (F134). This strategy was used for identification of substrates in synthetic peptide libraries in a probe of protein kinase substrate specificity using the known receptor tyrosine kinase V-ABL (F135). Results indicated that Ile is the optimal residue at the position N-terminal to tyrosine in a motif not previously identified to serve as a phosphorylation and autophosphorylation site. Other laboratories have suggested the identification of phosphopeptides by their dabsyl derivatives using capillary electrophoresis, with the subsequent involvement of electrospray mass spectrometry for characterization of the sites of phosphorylation (F136). A particularly interesting and careful piece of workconcerns the identificationof phosphocysteine,the catalytic intermediate of the mannitol transporter of the bacterial phosphotransferase system (F137). This study made effective useof tandem mass spectrometry for the sequence specific identification of the site of modification. A series of papers have used mass spectrometry in a variety of ways to identify the autophosphorylation sites of rhodopsin kinase (F138-F141). Clearly, collision-induced dissociation will play an ever-increasingrole in delineating phosphorylation sites on such phosphopeptides of appropriate size after prelimiary identification has been achieved by selected ion monitoring of the collisionally activated electrospray effluent. Other examplesof the use of tandem mass spectrometry include identificationof tyrosine-185as thesiteof autophosphorylation of recombinant mitogen-activated protein kinase (F142,F143). By use of continuous-flow FAB LC/MS, two proline-directed serine phosphorylation sites have been identified on phosphoproteinp19 (F144).Avariety of methods have been used to address phosphorylation of aB-crystallin on bovine lens (F145),bovine and porcine tau proteins (F146,F147), the HMGY protein from Lewis lung carcinoma (F148),the nervous tissue-specificprotein B-50(GAP-43) (F149),rat liver acetyl-coA carboxylase (F150),and the 80-kDa (MARCKS) protein kinase C substrate (F151). Hemoglobin Variants. This is a topic of routine clinical diagnostic importance at this juncture. Neonatal hemoglobin can readily be screened by electrospray measurement of the molecular weights of the a and @ chains directly. Mass shifts observed in proteolytic digestion and mass mapping are well suited to rapidly identifying the peptide(s) with modified sequences. These can then be sequenced in some cases by low-energy collision-induced dissociation and, if required, by high-energy collision-induced dissociation. This has been applied extensivelyby Shackleton and colleagues (F152,F153), and by Wada and colleagues (F154),and a large number of these have now been documented. Similarly, characterization of the products from site-directed mutagenesis in human hemoglobin may be established by these methods (FIJS),as well as confirmation of the production of unmodified adult hemoglobin expressed in Escherichia coli (F156).Shackleton, Smith, and co-workers have used collisional activation on the intact multiply charged human hemoglobin @-chainvariant proteins which differ by a single amino acid substitution (F157). In these experiments, they observed that the Thr 50-Pro 5 1 cleavage produced complementary y96 and bso sequence ions, as the most favored fragmentation pathway.
Not surprisingly, the Willamette variant form, in which Arg is substituted for Pro 5 1, does not shown preferential cleavage forming y96 product ions. From these data, these authors suggest that fragmentation differences in these collisioninduced spectra are substantially governed by primary rather than secondary structure. The application of molecular weight mapping of proteolytic or chemical digests of proteins forms the first level of mass spectrometric-based information. These results can be sufficient for identification in some cases and, if not, provide the basis for structural analysis of the components of a proteolytic digest to the necessary level of detail to arrive at a structurally unambiguous characterization of the protein. This strategy has been under continual modification throughout the last decade as new ionization techniques became available. Combinations of ionization techniques and mass analyzers, however, still have their pros and cons. A choice would probably be made on the basis of total amount of protein available for the problem at hand. If sufficient amounts of protein are readily available, mapping by liquid SIMS in connection with continuous-flow chromatographic inlet provides data with clearly identified correct nominal masses (F158). If sample is severely limited, as in spots from 2-D gel electrophoresis, matrix-assisted laser desorption ionization on the unseparated mixture may be attractive, despite the ambiguity of mass measurement (*1-2-Da range) without internal calibration. It is not always practical to use internal calibration with picomole level samples for MALDI (F159). If either low- or high-energy collision-induced dissociation will be required, at least on some components, then microbore liquid chromatography electrospray methodology is probably the method of choice (see relevant discussion in section D). Of course, as in the examples above using MALDI on digest mixtures, similar approaches can be taken using electrospray on digest mixtures, as illustrated by studies of the selective proteolytic accessibility of calmodulins in the presence of various concentrations of calcium ions (F160). The generation of molecular weight information on significant numbers of the components of a protein digest forms the basis for identification of the protein and any covalent modifications, as we have indicated repeatedly. Several groups have written specific computer algorithms to utilize this empirical molecular weight data to search existing protein and cDNA databases for possible matches to appropriate computer-calculated masses of expected digest products based on the protease or chemical cleavage reagent utilized in the experiment ( F 1 6 1 4 1 6 5 ) . While this sort of capability will eventually be a useful aid in initial data digestion and hypothesis generation, it is easy to think of a long list of false positives and false negatives which may result, particularly in the use of MALDI data, where the mass measurement accuracy does not provide nominal mass correctly in the original data set. Thus, while such strategies should be further developed for routine utilization in the process of characterization of proteins by mass spectrometry, it would be inappropriate to focus on such methods for unambiguous identification in the face of the ready availability of sequence information from Edman degradation and both low- and highenergy collision-induceddissociationspectra from tandem mass spectrometry.
Since there is such wide availability of sequence information from automated Edman sequencers, there is a strong impetus to developing methods to combine that information with molecular weight or sequence information from mass spectrometry. This integration of these types of data has been discussed in some detail by Johnson and Walsh (F166), including an algorithm which utilizes Edman data on peptide mixtures together with mass spectrometric data to make sequence assignment suggestions. Whether fortunate or not, there is a natural marriage between low-energy collisioninduced dissociation information and automated Edman information. The practical problems associated with both the Edman degradation and low-energy collision-induced dissociation would be obviated by use of high-energy collisioninduced dissociation for those aspects of protein identification where both unambiguous sequence and structural characterization are required (F9); nevertheless if limiting one’s strategy to the Edman and low-energy collision-induced dissociation intentionally, as Johnson and Walsh point out, both sets of data are required from the same sample to establish an unambiguous sequence. This situation worsens as minimization of sample consumption becomes an additional constraint. In that case, the absolute sensitivities from Edman sequencers and tandem quadrupole mass filters generate other problems, including the need to carry out one or two stages of chemical derivatization and repeated CID analysis to establish sequence. This situation is dramatically brought into focus with the pioneering work of Hunt and co-workers on the identification of antigenic peptides (F167-Fl69). Despite these caveats, Hunt and colleagues have recently identified a peptide recognized by five melanoma-specifichuman cytotoxic T-cell lines (FI 70). They estimate that this peptide was only present at five copies per cell, in a mixture extimated to contain at least 10 000 naturally processed peptides! The structures of other peptides associated with MHC class I molecules have recently been reviewed (FI 7 1 ) . A variety of protein sequence and structural problems have been reported using a combination of automated Edman degradation and mass spectrometric methods, including the observation of the occupied cobalaminbinding domain and characterization of the carboxy terminus of methionine synthase (FI 7 2 ) ,a major membrane lipoprotein of the Erdman strain of Mycobacterium tuberculosis (Fl73), primary structure of thioredoxin from Aspergillus nidulans (F174),and a truncated form of arrestin isolated from bovine rod outer segments (F175). Low-Energy Collision-InducedDissociation. Several protein sequences based on extensive use of low-energy collisioninduced dissociation have been reported. These include parvalbumin from cat, gerbil, and monkey skeletal muscle (F176,FI 7 7 ) ,phenylalanine hydroxylase-stimulating protein from rat and human liver (F178),6-pyruvoyl tetrahydropterin synthase from salmon liver (FI79),human red cell-type acid phosphatase, a cytoplasmic phosphotyrosyl protein phosphatase (F180),and inducible nitric oxide synthase from mouse macrophages (F181). Higb-Energy Collision-InducedDissociation. High-energy collision-induced dissociation spectra from four-sector mass spectrometers qualitatively contain three types of ions: immonium ions of many of the amino acids or covalently An&tical
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modified amino acids, which provide information on peptide amino acid composition (F182); similar suites of ions which may be attributed to low-energy processes (see section E for discussion);and side-chain and ring cleavage processesunique to experiments in which the deposition of internal energy is significantly higher. These latter processes are important in establishing amino acid sequence unambiguously solely from interpretation of the high-energy CID spectra, as in the case of the assignment of leucine vs isoleucine, the isobaric pair of amino acids. A recent discussion of the interpretation and utilization of high-energy spectra has been prepared (F183). Further work on the optimization of sensitivity includes the use of microbore LC with continuous liquid SIMS introduction and rapid-scanning multichannel array detection based on readout of the multichannel array with a charge-coupled device in real time (F184). Use of continuous-flow inlet with CID spectrum recording times of approximately 10 s minimizes component suppression effects (F185) and permits on-line liquid chromatography high-energy CID mass spectral analysis of protein digests (F186). The use of electrospray ionization in connection with four-sector high-energy collision-induced dissociation shows considerable promise, as indicated by Fenselau and coworkers (F187), showing high-energy CID spectra at a significantly higher mass range for doubly protonated speciesthan is currently routine using singly protonated species from liquid SIMS ion sources. The nature of high-energy CID spectra from chymotrypticdigests of proteins which have been acetylated prior to digestion has been reported (F188). An interesting procedure for the partial C-terminal hydrolysis of peptides and proteins has been reported using 90% pentafluoropropionic acid or heptafluorobutyric acid at 90 OC for various time periods, followed by mixture analysis with either liquid SIMS or electrospray ionization (F189). It is worthwhile reiterating the report on the correction of the molecular weight of myoglobin, a common calibrant for MALDI and electrospray techniques (FI 90). Papayannopoulos and Biemann have reported the utility of cysteine cyanylation in chemical cleavages of protease inhibitor isolated from the hemolymph of Sarcophaga bullata larvae (F191). The high-energy CID spectra of the resulting N-terminal cycliccysteinepeptides are presented (F191). This methodology continues to be used in the determination of primary structure of a variety of proteins, including thioltransferase (glutaredoxin) from human red blood cells (F193); Gag proteins from the MN strain of HIV virus type 1 (FI 93); the primary structure of Gal@l,3(4)GlcNAca2,3-sialyltransferase (FI 94); bovine serum amine oxidase (F195), adenosinephosphopeptide inhibitors ( F196); the unusual 0-acylation of serine-4 by biotin of gonadotropin-releasinghormone(F197); proteins in the yeast signal recognition particle (FI98); gastrinreleasing peptide (FI 99); soluble lactose-binding lectin from Xenopus laeuis skin (F200); solublelactose-bindinglectin from rat intestine bearing two carbohydrate-binding domains on the same protein (F201); another solublelactose-bindinglectin, L-29, of rat and dog bearing a Pro-Gly-Tyr-rich bacterial and tissue collagenase-association domain (F202); microbial transglutaminase from Streptoverticillium sp. strain s-8 112 688R
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(F203); an artifact of Alzheimer’s amyloid @-peptide(F204); and palmitylation of a G-protein coupled receptor (F205). Protein Covalent Modification. Residue asparagine-57 of bovine liver cytochrome b~ was replaced with a cysteine residue and expressed in E. coli. A mixture of four major specieswas obtained, including heme covalentlybound through a thioether linkage involving cysteine-57 and the a-carbon of the heme 4-vinyl group, covalently bound heme coupled through a thioether linkage involving the @-carbonof the heme 4-vinyl group, an analogous thioether linkage oxidized to the sulfoxide, and a green form with spectroscopic properties characteristic of a chlorin (F206). Electrospray mass spectrometry and electronic spectroscopy, proton NMR, Raman spectroscopy, and electrochemistry were required to establish the nature of the four species. A very interesting paper on the multiplicity of binding of protoporphyrin IX to up to three protein side chains has been reported from studies of the active site heme P460 in hydroxyamine oxidoreductaseof Nitrosomonas (F207). Electrospray ionization mass spectrometry was used to probe the nature of the leucocyte elastase @-lactam-derivedE-I complex (F2084212). A similar utilization of electrospray resulted in detection of transient intermediates in the Wittig, Mitsunobu, and Staudinger reactions directly from solution (F213). Further work on the interaction between hen egg white lysozyme and tri-N-acetylglucosaminehas been reported using chemical modifications,digestions,and electrospray mass spectrometry (F214, F215). The primary structure and location of a lipoic acid residue from H-protein of Pisum satiuum has been reported (F216). The covalent binding of phase I1 acyl glucuronides of certain nonsteroidal antiinflammatory drugs has been shown to occur through an imine mechanism, using high-energyCID analysis (F217). A similar strategy was utilized to establish the alkylated peptide between melphalan and the protein metallothionein (F218). Carbodiimide was used to catalyze intramolecular cross-linked protein studying the association of interleukin-l@(F219). Further work on chemical modification of interleukin- 18using acrylodan showed modification of both cysteine-8 and lysine103 residues (F220). A report of specific glycation of lens crystallins by ascorbic acid has appeared (F221). Attachment sites of superoxide dismutase derivatized by poly(ethy1ene glycol) have been established (F222, F223). Work on the low-affinity metal-binding site in the light chain of tetanus toxin has been reported, involving a modified histidine residue (F224). Irreversible inactivation of mouse ornithine decarboxylase has been studied using a-difluoromethylornithine (F225). Epoxide-based inhibitors have been shown to bind to the active site of Bacillus amyloliquefaciens 1,3-1,4-@-~-glucan4-glucanohydrolase at glutamic acid 105 (F226). Work on the structure-function relations in mutagenized human aromatase has shown that the enzyme is glycosylated (F227). Localization of the active site serine in prolyl endopeptidase from Flavobacterium meningosepticum has been established by electrospray mass spectrometry (F228). Mechanism-based inactivation of leukotriene 4hydrolase/aminopeptidase has been characterized using leukotriene A4 (F229). The active site peptides from 2-ethynylnaphthalene-inactivated cytochrome P450 2B1 and 2B4 has been reported (F230), using
matrix-assisted laser desorption mapping of the modified peptides. Use of immobilization of cytochrome P450 on a gold-plated surface permitted the washing away of contaminants with water to circumvent the cumbersome and timeconsuming alternative procedure involving electroblotting onto PVDF membranes. Use of albumin as an internal mass calibration standard was necessary to bring the mass accuracy below 0.1% error. In addition, it was noted that 100 mM phosphate buffer suppressed the signal of these proteins under MALDI conditions, and even a dilution of 20 mM required higher laser power and the associated photochemical adduct formation and broadened peaks gave poor accuracy of mass measurement (F231). Studies designed to probe the close association of actin monomers were carried out using a bifunctional cross-linking reagent N-(4-azidobenzoyl)putrescine. After tryptic digestion, sequence analysis and mass spectrometry were used to establish that the cross-link was between glutamine-41 on one monomer and lysine-1 13 on another monomer (F232). Studies aimed at gaining information on surface topology (F233)and protein interface topology (F234) have been probed by a combination of acetylation and mass spectrometric identification of the modified residues. Gel Electrophoresis. In a recent review of the 2-D gel protein database on human keratinocytes, it is noted that only a small proportion of the total set of proteins have been identified and that little is known about the composition of basal and differentiated cell types. In addition, a plea is made todevelop strategies to integrate protein and DNA information to generate comprehensive approaches to the study of cell types (F235). There are now a number of reports involving various ways that new methods of mass spectrometry might be employed to assist in the characterization of proteins isolated from 1or 2-D gel electrophoresis. They include direct measurement of the molecular weight of proteins isolated from the gels, mass mapping of proteolytic digests of such proteins, and tandem mass spectrometric sequencing of these proteins. In general terms, proteins may be electroblotted onto some sort of solid matrix for measurement of molecular weight directly and for subsequent in situ digestion and measurement of the molecular weight of proteolytic digest components. Alternatively, they may be electroeluted into a buffer for similar purposes. There should be advantages to the membrane blotting strategy which would accrue from detergent and buffer salt removal by simply washing, while the electroeluted protein would require either acetone precipitation for removal of Coomassie stains and residual SDS or some equally effective alternative procedure. A variety of membranes have been employed, including nitrocellulose (F236), poly(viny1idine difluoride) and polyamide membranes (F237-F239), and nylon (F240). As an alternative, digestion of the protein in situ in the gel has been carried out successfully,both for analysis of recombinant proteins at the nanomole level (F241) and for the sequence identification and cloning of proteins at the picomole level (F198). Use of electroelution and sequence determination by high-energy CID analysis has been illustrated in the identification of a number of proteins from human melanoma cell culture lysates (F242, F243).
Noncovelent Associations. One of the more intriguing attributes of the electrosptay ionization process is its ability to sample noncovalent solution associations intact in the gas phase. This work has generated some skepticism and controversy on the topic of how to determine that the noncovalent association observed by mass is representative of specific interactions established by methods in solution such as NMR. Nevertheless, this is an interesting and exciting topic which might provide a completely new experimental approach to gaining information about protein folding intermediates, particularly in combination with proton vs deuterium exchange. Some recent examples are the observation of tetrameric forms of concanavalin A, which may be disrupted by either heating or collisional activation in the electrospray interface (F244). Since the amount of internal energy may be a complex function of electrospray ion source operating parameters, it was important to show that the intact heme-bound myoglobin species could be detected by an electrospray ion source in a double-focusing mass spectrometer (F245). Other examples include the study of noncovalent dimers of leucine zipper peptides (F246)and the stoichiometry of the T4 gene 45 protein (F247). Detection of transient protein folding populations by hydrogen/deuterium exchange and NMR experiments are being explored in detail (F248-F251). In addition, hydrogen/ deuterium exchange strategies have been suggested as ways of obtaining information on the nature of fragmentation processes (F252)and as tools for protein structure elucidation (F253, F254). Metal Ion Associations. Further work aimed at defining peptide protein metal ion recognition sequences has been reported, including the copper-binding sites of histidine-rich glycoprotein (F255, F256) and the ability of zinc finger domains to bind copper ions in the case of the human estrogen receptor protein (F257, F258). In addition, by combining pH control with electrospray mass spectrometry, the ability of reconstituted metallothioneins to bind up to seven metals has been reported (F259). In this study, it was clear that the number and types of cations incorporated per molecule of protein may be readily measured directly in this experiment. Studies of the zinc-binding properties of the synthetic nucleocapsid protein in cp7 of HIV-1 has been reported (F260). The discovery of an extremely tight calcium-binding site in the peptide Met-A1 8-B28 from the plasma membrane calcium pump was revealed by detection of 39 additional mass units in the electrospray mass spectrum. Other sites in this protein have also been studied, and it is suggested that these extremely tight EDTA-insensitive sites are probably consitutive binding sites in the protein (F261). A rather interesting suiteof experimentsinvolving calciumdependent association of the Lewis X glycolipid has been reported using electrospray methodology with collisional activation to probe the binding affinity (F262). These results suggest a predilection of Lewis X homotypic interactions, which may correlate with the biological utility of this cell surface carbohydrate in the cell adhesion process.
0. CARBOHYDRATES AND GLYCOCONJUGATES As is true for studies on other biopolymers, the electrospray and MALD ionization methods are particularly well suited Anaiyfcai Chemistty, Vol. 66,
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to provide a global view of labile heterobiopolymericsubstances intact. Hence, it is now possible to acertain exactly the empirical moiety composition for each discreet molecular species comprising a “microheterogeneous” mixture. Therefore, it is also possible to obtain a precise framework for establishing which components go togther. Presently, the electrospray method, even with quadrupole mass analyzers, is able to provide superior mass resolution for such purposes. With the advent of electrospray ionization adapted to sector instruments with multichannel array detection, this method will clearly excel in both mass resolution and mass measurement accuracy while preserving at least comparablesensitivity. While this latter capability has not become available in many laboratories as yet, it represents a major advance in obtaining the most precise compositional framework for low charge state, higher mass components within which all other data obtained from characterization of degradation products must fit. Simultaneously, the usually encountered global microheterogeneity is obtained directly from such a molecular weight profile on the chemically unseparated “natural” mixture. Thus the gross compositional integrity of the mixture of closely related structures is in hand. Of course, just the isolation of the sample might introduce unsuspected alterations of labile functions or moieties which must still be dealt with eventually. However, of critical importance is the ability of the technique to operate in a truly “soft” manner if used properly. First one must have detectability. While SDS-PAGE or CZE may reveal heterogeneity, mass analysis provides the ultimate authority. Recently it has become clear that MALDI is not such a soft ionization technique per se. Indeed, a significant fraction of molecular species possess high internal energy (“natural” metastable ions). Analytical advantage may be taken of decomposition product ions using reflectron stepping timeof-flight analysis to record their mass spectra ( F l l d ) . In addition to the global view, very powerful methods are currently widely employed in structural characterization of carbohydrates released from glycoproteins and glycolipids. Unlike the usual approach to proteins, protocols for dealing with biopolymers consisting of relatively large carbohydrate moieties involve chemical derivatization in their strategies to effect cleanup from biological matrices and salts (especially nonvolatile buffers), determine monosaccharidecomposition and linkage (methylation analysis), counteract hydrophilicity of the oligosaccharide, and enhance sensitivityof mass spectral detection, direct fragmentation, and so on. Where tandem mass spectrometryhas been employed, it has provided a wealth of structural information directly. Several reviews and book chapters have become available covering various aspects of this very complex, challenging field: carbohydrate analysis (GI,G2), oligosaccharides (G3-G5), glycoproteins (G6, G7), glycolipids and lipoproteoglycans (G7), oligosaccharides that mediate cell adhesion (G8), 0-linked fucose (G9), gangliosides (GIO),glycolipids (GII), and glycoconjugates (GI2). The uninitiated reader should bear in mind that there is an integrative quality to these fundamental reviews which requires perusal of the previous review (BI)to gain a balanced picture of the context of this field briefly, as noted above. The new techniques provide information about higher levels of 668R
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molecular organization noted above, but the strategies for dealing with the structural characterization of the individual component moiety classes are well described in the previous review (BI), and little new or remarkable has been added. This ability to globalize is most apparent in the discussion of molecular weight measurements and profiles in the glycoprotein portion of section F and in the ability to detect larger heterooligomeric species of glycolipids by application of the same soft ionization techniques. Derivatization adTechdques. Methylation is widely used as a starting derivative for composition and linkage analysis of the subsequent total hydrolysate, as illustrated by the detection of 6-O[(R)- 1-carboxyethyll- galactose as a component of the extracellular polysaccharidefrom Butyriuibrio fibrisoluens strain X6C61 ( G l 3 ) . One common alternative strategy is direct hydrolysis of the oligosaccharide, followed by formation of the methyl glycoside or ester, in the case of neuraminic acid derivatives, and then analysis by electron impact mass spectrometry on the trimethylsilylated ethers. One example of this approach concerns the characterization of the incorporation of chemically synthesized N-propanoylDglucosamine or N-propanoyl-Dmannosamineinto N-acetylneuraminic acid in the rat in vivo (GI4). Interest continues in studying the information content of mass spectra using small oligosaccharidesof all possible linkage isomers. Using LSIMS with Fourier transform mass spectrometry, the oligosaccharide anions of a variety of the trisaccharides have been studied (CIS). Using similar ionization, the peracetylated analogues of small oligosaccharides have been studied by CID techniques (GI@. Further work on direct chemical ionization of polysaccharides has continued in the mass range up to several thousand daltons (GI7). The general procedure for the release of intact nonreduced forms of both N- and 0-linked oligosaccharides from glycoproteins by chemical cleavage with hydrazine has been described in some detail (GI8). Work on a centrifugal sizeexclusion chromatographic method for rapid desalting of carbohydrate samples prior to liquid SIMS has been reported (GI9). Initial reports on the use of electrospray and lowenergy collision-induced dissociation for the characterization of N-linked oligosaccharides released by digestion with PNGase F has been described (G2O). Further work on the oligosaccharides from hyaluronic acid and chondroitin 6-sulfate by negative electrospray mass spectrometry has been explored (G2I). Work on MALDI TOF for a variety of oligosaccharides has been carried out in the context of interest in quantitative analysis (G22). Among the matrices studies, 2,5-dihydroxybenzoic acid gave detection limits in the 100-fmol range. Derivatization of these oligosaccharides by peracetylation increased this detection limit by a factor of 10(G22). MALDI was used for the analysis of glycopeptides derived from each glycosylation site of the &subunit of (Na,K)-ATPase (G23). In addition to permethylation, reductive amination of free reducing termini of oligosaccharides has become an important oligosaccharide derivatization protocol, which has the advantage of leaving the glycan free for endoglycosidicdigestion and further analysis. Use of the p-aminobenzoic acid ethyl ester for study of caprine p-mannosidosis with high-energy
collisional activation has been reported (G24, G25). Further work using the p-aminobenzoic acid butyl ester and negative ion collision-induced dissociation mass spectrometry reveals some linkage positions (G26). Reductive amination with n-hexylamine followed by permethylation has been used to characterize the oligosaccharides on baculovirus-expressed mouse interleukin-3 (G27, G28). A further variation involves reductive amination with octylamine and subsequent Nalkylation with 2,4-dinitrofluorobenzene(G29). The use of 1-(4-methoxy) phenyl-3-methyl- 5-pyrazolone has been studied for the derivatization of the reducing terminus of hyaluronic acid (G30). LC-electrospray analysis, using oligosaccharides reductively aminated with 2-aminopyridine, was reported (G3Z). The pyridylamino sugar derivative may be converted to a 1-amino-1-deoxy derivative for subsequent labeling with fluorescein or biotin (G32). Specificity of mild periodate oxidation of oligosaccharide alditols was probed using 1,2dipalmitoyl-sn-glycero-3-phosphoethanolamine (G33). Oligosaccharides. Studies of the carbohydrate structures of a wide variety of glycoproteins have involved permethylation and liquid SIMS analysis. Several examples have utilized this methodology effectively,usually in conjunction with proton magnetic resonance analysis. Studies of a thrombin-like serine protease from the venom of the Malayan pit viper, Agkistrodon rhodostoma reveal partially truncated di-, tri-, and tetraantennary complex-type N-glycans, and solely (a2-3)-linked sialic acid substituents (G34). Structural studies of the 0-linked glycans occurring on mucins from a variety of sources have been reported, including those from human amniotic fluid (G35), human colonic cancer cell line CL.16E (G36), and bovine submaxillary glands (G37, G38). The Nglycosylation of both human transferrin (G39) and human transferrin receptor (G40) has been reported. Characterization of the N-glycans of nonsecretory ribonucleases from kidney, liver, and spleen have been detected by electrospray mass spectrometry after PNGase Fliberation (G41). A novel terminal element has been rivealed in human urokinase containing GalNAcP( 1-4) [Fuca( 1-3)]GlcNAB( 1-2) (G42). Structural studies of human milk oligosaccharides have provided evidence for a new core structure, the p-lacto-Noctaose (G43, G44). Characterization of the sialylated oligosaccharides of human erythropoietin expressed in recombinant BHK-21 cells has been carried out in considerable detail (G45). This study is illustrative of the continuing difficulty of interlaboratory comparison of the analyses of oligosaccharides on such a complex glycoprotein by somewhat different analytical strategies. Characterization of the 0linked oligosaccharides on glycoproteins GP I and G P I1 of Fusarium sp. M7-1 has been reported (G46-G48). The complexity of oligosaccharide alditols released from one site of a mucin from rat small intestine was illustrated by GC/MS analysis of the permethylated neutral fraction (G49). Together with the sialic acid-containing species, 29 components were detected without characterization of sulfate-containing components. In studies of bovine pro-opiomelanocortin, the Lewis X epitope was found to be a major nonreducing structure in the sulfated N-glycans (G50). The N-linked oligosaccharides of frog (Rana pipiens) rhodopsin have been characterized by permethylation and electrospray mass spectrometry (G5Z).
In studies of the marine sponge Microciona prolifera, a novel pyruvylated carbohydrate unit has been identified which is implicated in cell aggregation (G52). A carbohydrate-linked 2-(aminoethy1)phosphonate has been characterized as a consitituent of an apolipophorin I11 from the insect Locusta migratoria (G53). From studies of a carbohydrate-rich glycopeptide isolated from the fertilized eggs of Indian Medaka fish, Oryzias melastigma, a novel type of tetraantennary sialoglycan has been described (G54), belonging to the L-hyosophorin family. Sulfated Oligosaccharides. One of the most interesting and important pieces of work (by Lawson, Feizi, and colleagues) was the discovery of a sulfate-containing fucotetrasaccharide with high affinity for the human E-selectin molecule (G55). This is a new class of oligosaccharideligand for human E-selectin present among oligosaccharides from ovarian cystadenoma glycoprotein. The oligosaccharides from the glycoprotein were released by mild alkaline base elimation, conjugated to lipid, resolved on thin-layer chromatography plates, and tested for binding by Chinese hamster ovary cells transfected to express E-selectin. Liquid SIMS analysis of the E-selectin binding neoglycolipid fraction on the TLC plate showed the preparation to be an equimolar mixture of the Lea and Lex/SSEA- 1-type fucotetrasaccharidesulfated at position 3 or the outer galactose. Sulfated sialyl oligosaccharides have been identified in trachiobronchial mucus glycoproteins from a patient suffering from cystic fibrosis (G56). A new saccharide structure having a sulfated fucose as an internal residue has been isolated and identified from the jelly coat of echinoderm eggs in the acrosome reaction-inducing substance (G57). Several reports have been concerned with the characterization of the sulfated components of heparin-derived oligosaccharides (G58-G6Z). One of these describes the liquid SIMS behavior of acetylated derivatives of sulfated oligosaccharides derived from heparin and discusses the stability of sulfategroups to base-catalyzed acetylation (G6Z). Sulfated and nonsulfated pentameric oligomers have been identified as nodulation factors from Rhizobium tropici (G62). Periodate Oxidation. Further work has been reported using the periodate oxidation of vicinal glycols followed by reduction with borohydride and derivatization by either acetylation or methylation by Nilsson and co-workers (G63, G64). This procedure has the advantage of revealing residue linkages when this derivative is analyzed by liquid SIMS. Application to the analysis of 0-linked oligosaccharides from human glycophorin A illustrates the analysis of the peracetates by GC/MS as well as liquid SIMS (G63). The method has also been applied to characterization of the structural heterogeneity of the carbohydrates isolated from a mouse monoclonal immunoglobulin A antibody (G64). In many cases, the molecular weight distributions of intact glycoproteins can be determined by electrospray mass spectrometry. Some work has been devoted to exploring the usefulness of this information for quality control of recombinant proteins, and the so-called "antennicity" obtained from electrospray molecular weight relative abundances has been correlated with information on the same recombinant protein by HPLC and NMR results (FZ22, G65, G66). This has been illustrated using chimeric glycoprotein K2tuPA (F65). Analytical Chemistry, Vol. 66, No. 12, June 15, 1994
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Similar molecular weight profiles obtained from electrospray can and should be correlated with results from analysis of the nature and composition of the oligosaccharides on the same glycoprotein. Mass heterogeneity on the F, part of two mouse IgG monoclonal antibodies, M N 12 and WT3 1, have been resolved by electrospray mass spectrometry (G67). Electrospray mass spectrometry was used successfully for the analysis of the C-reactive protein subunit composition from Limulus polyphemus (G66). Nine components were detected, four of which corresponded precisely to previously reported protein sequences glycosylated by a single oligosaccharide highmannose chain. Lipopolysaccharides/Lipooligosaccharides. These complex endotoxins are comprised of a membrane-anchoring lipodisaccharide called “lipid A” linked through a labile ketooctulasonic acid (KDO) to a “core” oligosaccharide, together termed lipopolysaccharide, and in many cases futher elaborated by a repeating carbohydrate antigenic unit. Mild acid hydrolysis liberates lipid A from the rest of the carbohydrate oligomeric elaboration. Lipid A has been studied in some detail from a variety of Gram-negative bacterial pathogens. A recent report on the comparison of lipid A preparations from Enterobacter agglomerans, commonly found in cotton and cotton dust, has been compared with that from Salmonella minnesota by plasma desorption mass spectrometry (G68). The use of PDMS on lipid A from E. coli and Escherichia hermani, together with Salmonella from a variety of strains, has been reported (G69). In addition, negative ion liquid SIMS has been used to investigate the ssc permeability mutant of Salmonella typhimurium (G70). One of the impressive achievements during this review period has been development of the ability to analyze the oligosaccharide portions from lipopolysaccharides by both plasma desorption mass spectrometry (G71) and electrospray mass spectrometry (G72). With these capabilities in hand, one can be assured that the nature and diversity of the labile functionality associated with these endotoxins may be properly detected and structurally described. One of the success stories of modern mass spectrometry has been the structural detail with which the major oligosaccharides from a variety of Gramnegative bacteria have been characterized, revealing a suite of host cell surface glycosphingolipid mimicry which is now thought to protect these human pathogens from immune surveillance. The primary tools have again been methylation analysis, high-energy collision-induced dissociationusing highperformance tandem mass spectrometry, LC-electrospray mass spectrometry, and the old standby, liquid SIMS. Work on the major oligosaccharides from a variety of these organisms has been described, including the major lipooligosaccharide from a strain of Haemophilus ducreyi, the causitive agent of chancroid, a genital ulcer disease (G73), the cell surface lipooligosaccharides from a nontypable strain of Haemophilus influenzae (G74), strain AH1-3 (G75), and H. influenzae type b strain A2 (G76). Based on analogous earlier structural studiesof pathogenic Neisseria, the first step in the biosynthesis of this lipooligosaccharide has been taken by the cloning, identification,and characterization of the phosphoglucomutase gene (G77). In an extensive series of reports, the chemical structures of Campylobacter jejuni low molecular weight lipopolysac670R
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charides (viz. lipooligosaccharides) have been established, associated with some members of the seriologically diverse reference strains (G78-G85). This species (C. jejuni) is recognized as a leading cause of human enteritis, and the serotyping thus far includes 48 different isolates of this species. More recently, there are clinical indications that the GuillaimBarr6 syndrome, the most common cause of acute general paralysiswhich usually occurs one to three weeks after bacterial or viral infection, follows a previous C.jejuni infection (G85). Thus far, serotypes 0:1, 0:4,0:23, 0:36,0:2, 0:30, and 0:19 have been structurally examined (G78-G85). One of the intriguing results of this extensive work is that the lipopolysaccharides provide another example of structural variation between strains, very similar to that found in the low molecular weight LPS core lipooligosaccharides of the Neisseria and Haemophilus species pioneered by Griffiss and colleagues. In this work, the authors point out that the most striking feature of the core oligosaccharide structures from C.jejuni LPS is their structural similarity with glycosphingolipids and gangliosides. As indicated above, this degree of molecular mimicry seen initially in Neisseria gonorrhoeae (G86) is thought to play a major role in the virulence of these human pathogens by providing a means of evading the immune response of the host to the organism’s infection (G79, G86). Studies of the lipooligosaccharide antigen from Mycobacterium gastri have revealed a molecular marker permitting unambiguous differentiation of M . gastri from Mycobacterium kansasii, two strains which have long been considered by some bacteriologists as synonymous. This is important, since M. kansasii is the known etiologic agent of pulmonary disease, while M. gastri is considered nonpathogenic (G88). The complete structure is a C4-branched 3,6-dideoxy-cu-xylohexopyranose. Studies of the trehalose-containing lipooligosaccharides of Mycobacterium gordonae have been aimed at distinguishing this seropathogen from Mycobacterium tuberculosis (G89). M. gordonae can cause pulmonary disease indistinguishable from that caused by M. tuberculosis, and patients often are started on antituberculosis therapy, yet M . gordonae is resistant to normally indicated treatment. This work will now permit unequivocal identification of M. gordonae, an opportunistic pathogen of increasing frequency of occurrence in patients with AIDS. Further studies of the 0-antigenic polysaccharides include characterization of a lipopolysaccharide of Vibrio cholerae strain H11 (non-01), which is an important human pathogen causing diarrhea, and possibly death, if not treated appropriately (G90). The lipopolysaccharide of the recombinant strain E. coli F5 15-207 was used to express and obtain genus-specificepitope of Chlamydia lipopolysaccharide (G91). Structural elucidation of the pure oligosaccharide is now available for serological experiments designed to characterize the specificities of monoclonal and polyclonal antibodies against chlamydial lipopolysaccharide. A variety of other antigenic polysaccharides have been studied, including sialic acid-containing E. coli 0104 lipopolysaccharide (G92), the structural analogues of group I1 K antigens in E. coli isolated from Rhizobium fredii and Rhizobium meliloti (G93), the lipooligosaccharide nodulation signals produced by type I and I1 strains of Bradyrhizobium japonicum (G94), the exopolysaccharide produced by Lactococcus lactis subspecies
cremoris H414 (G95),the Lactobacillus delbriickiisubspecies bulgaricus rr (G96), and the lipopolysaccharide of E. coli rough mutant F653 representing the R3 core type (G97). Glycopeptidolipid SurfaceAntigens. In continuing studies of the kinetics, biosynthesis, and roles of the dominant surface glycopeptidolipid antigens of Mycobacterium avium in pathogenesis, two rough mutants, Rg-0 and Rg-1, have been discovered which contain two novel lipopeptides devoid of the carbohydrate substituents which confer serotypic activity on the previously identified glycopeptidolipid antigens (G98). These newly discovered lipopeptides are identical to the fatty acyl-tripeptide-amino alcohol core component of the glycopeptidolipids of the M. avium complex. This discovery will be a major aid in the elucidation of the biosynthetic pathway of GPLs, and the means to examine the roles of GPLs in the disease process induced by M. avium, in particular in eliciting an immunosuppressive response. Studies of the structural nature of the glycopeptidolipids from Mycobacterium xenopi have been reported (G99). Matrix-assisted laser desorption has been employed to reveal the more global nature of a mycobacterial lipoarabinomannan from Mycobacterium bovis BCG (G100). The multiglycosylated form of the mycobacterial mannosyl phosphatidylinositol has been described (GIOI). The structure of a novel sulfate-containing mycobacterial glycopeptidolipid has been established by liquid SIMS and 2-D N M R (G102). The peptidoglycan composition of a highly methicillin-resistant Staphylococcus aureus strain has been reported (G103), together with similar studies of a hetergeneous Tn551 mutant of this strain (G104). The structure of the H.influenzae peptidoglycan has been established and compared with the composition of the peptidoglycan from ampicillin-resistant and ampicillin-sensitive strains (G105). The muropeptides from E. coli murein sacculus have been studied (G106). Structural studies of pneumococcal lipoteichoic acid have been reported by established mass spectrometric methodology, and in addition, matrix-assisted laser desorption mass spectrometry was employed to obtain a more global view of the heterooligomer composition intact (G107, G108). The 0specific polysaccharide component of the lipopolysaccharide produced by Fusobacterium necrophorum has been shown to be of the teichoic acid type, using liquid SIMS and linked scanning (G109). In addition, the capsular antigen of E. coli is of the teichoic acid type, and the structure of the repeating units has been established (GIIO). Structural characterization of the mannose cap lipoarabinomannan from Mycobacterium tuberculosis has been carried out on the permethylated oligoalditols by liquid SIMS ( G I I I ) . Further work on the structure of the lipophosphoglycan from the protozoan parasite Leishmania donovani has been reported (GI 12). In addition, structural studies have characterized Leishmania mexicana lipophosphoglycan (GI 13) and promastigotes (GI 1 4 ) . The complete primary structure of Leishmania major amastigote lipophosphoglycan is like the promastigote of all Leishmania species studied, composed of a PI-lipid anchor, a phosphorylated hexasaccharide core, phosphorylated repeats, and a neutral cap (G115). Glycosphingolipids. For more than twenty years mass spectrometry has played a key role in the structural characterization of glycosphingolipids. These widely diverse car-
bohydrate structures have begun to find their role in recognition and function, particularly as antigenic substances in cell surface recognition receptors for bacteria. Mass spectrometry has been employed to characterize the product of incorporation of 2-deoxy-galactose into gangliosides in vivo (G116). An improved preparation method for negative ion liquid SIMS of lysogangliosides has been reported (GI 17). Karlsson and co-workers have reported a new method for the isolation of polyglycosylceramides from human erythrocyte membranes (G118). The procedure can be used for large- and smallscale preparations of complex glycosphingolipids and has proven especially suitable for screening for polyglycosylceramides in different tissues. A membrane-bound endoglycoceramidase from Corynebacterium sp. has been characterized (G119). Useof alkali-metal attachmentwithcollisioninduced dissociation has been explored using glycosylceramide structural standards (G120). A highly polar matrix for negative ion liquid SIMS of native gangliosides has been suggested (G121). Conditions for obtaining MALDI spectra of underivatized and permethylated gangliosides have been discussed in some detail (G122). The authors conclude that the sensitivity of MALD ionization for molecular weight determinations is at least 2 orders of magnitude better than liquid SIMS FAB and that the derivatized gangliosides were ionized more efficiently in the negative ion mode. Further examples of MALDI have detected the blood group glycosphingolipids up to 40 monosaccharide units (G123). Structural studies of the glycosphingolipids have been reported from a number of organisms, including eggs of the sea hare, Aplysia juliana (G124); the nervous system of Aplysia kurodai (G125);the eggs of the sea urchin, Hemicentrotus pulcherrimus (G126, G127); the rainbow trout ovarian fluid (GJ28); the fox tapeworm, Taenia crassiceps (G129); Trypanosoma cruzi (G130); metacestodes of the parasite Echinococcus multilocularis (GI 31); and the amastigote forms of Leishmania amazonensis (GI 32). Monosulfated isoglobotetraosylceramide has been identified in rat kidney (G133). Two new decaglycosylceramides with blood group A-active tetrasaccharide repeats have been found in the epithelial cells of the small intestine of inbred rats (GI 34). An interesting disialoganglioside has been characterized from the lymphocytes of rat spleen (G135). Studies of the gangliosides from resting and endotoxinstimulated murine B lymphocytes have revealed the predominance of the G M pathway, ~ ~ wherease T lymphocytes and macrophages show a predominance of the GMlb type ganglioside expression (GI 36-GI 38). 0-Acetylated gangliosides have been isolated and characterized from bovine buttermilk (G139). A new ganglioside antigen termed Cholla-P has been identified in bovine brain which is recognized by the cholinergic neuron-specific antibody, Chol-la! (G140). Discussion of the structural complexity of antigens within the A B 0 blood group system has been presented in the context of graft survival and ABO-incompatible kidney transplants (G141). The occurrenceof novel I-typeglycolipid with sialylT6Gal structure has been characterized in human hepatoma (G142). The characterization of neutral glycosphingolipids has been carried out in the human cataractous lens (G143), human myeloid cells (GI&), human meconium (G145),and Analytical Chemistty, Vol. 66,No. 12, June 15, 1994
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the rare blood group p phenotype (G146, G147). Long-chain cyclic acetal modification of glycosphingolipids forms substances called plasmalocerebrosides. Identification of these structures has been reported (G148, G149). Human colonic adenocarcinoma cell line Colo205 was shown to contain the trifucosyl-Leb antigen (G150). A novel repetitive blood group A heptaglycosylceramide has been characterized by highenergy CID analysis (G151).
H. MISCELLANEOUS APPLICATIONS I N BIOLOGICAL RESEARCH The breadth of application of mass spectrometry to problems of structural characterization and quantitative analysis defies comprehensive review. Accordingly, we set quite limited aims in this section, namely, to highlight a few examples of recent research which illustrate the innovative application of relatively new developments in mass spectrometric methodology to problems of biological significance. In particular, we focus on the use of tandem mass spectrometry in the qualitative and quantitative analysis of compounds of biological and clinical relevance, with particular reference to the application of analytical modes other than product ion scanning, which is so widely used in the characterization of biopolymers. In addition, we refer to some applications of liquid chromatography/mass spectrometry, particularly using atmospheric pressure ionization sources, to supplement the coverage of this topic in section D. An excellent example of the application of advanced mass spectrometric techniques to clinical research is the continuing workof Millington and his colleagues ( H I ,H2) on the analysis of newborn blood for abnormal acylcarnitine and amino acid profiles. The objective of this approach is the development of procedures applicable to the screening of very large numbers of samples, necessitating the implementation of automated techniques for sample preparation and introduction to the mass spectrometer. The status of this program has been reviewed recently ( H 3 ) . The procedure involves extraction of blood spots on filter paper and esterification prior to solution in methanol-containing glycerol matrix and a surfactant. Analysis is by continuous-flow LSIMS. Esterification has been demonstrated to result in substantially enhanced sensitivity of detection for the acylcarnitines, with further improvement associated with the presence of surfactant; concentrations of 40.1 pM are detectable (H3). Highly selective detection of acyl carnitines is achieved by tandem mass spectrometry using a triple quadrupole instrument. Scanning of precursors of m / z 85 yields a profile of acylcarnitines, analyzed as the butyl esters; constant neutral loss scanning of 102 u gives selective detection of amino acid butyl esters ( H I , H2). Rashed and co-workers ( H 4 ) recently reported a similar approach to the analysis of acylcarnitines in blood spots but using electrospray ionization in place of LSIMS; detection limits were not reported but the approach was successful in comparing normal subjects and those suffering from inherited metabolic diseases. These authors ( H 4 ) suggested that electrospray sample introduction/ionization may be more amenable than continuous-flow LSIMS to the repetitivesample introduction required of screening studies. Multiple-scan modes in tandem mass spectrometry have also been applied to the characterization of phospholipids. Analyses using product ion scanning have been reported using 672R
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four-sector (H5,Hd), triple quadrupole (H7-HI 1), and hybrid (H12) instruments. Bryant et al. (H5, H6) employed sequential MS (MS3) on a four-sector instrument to characterize the sites of unsaturation in the acyl substituents, based on charge-remote cleavages of the carboxylate anions. Much attention has been given to the tandem mass spectrometric differentiation of isomeric glycerophosphocholines which incorporatethe sameacyl substituents but at different positions (snl and sn2). Huang et al. (H13) have reviewed some of the early studies. Kayganich and Murphy ( H 7 ) concluded that CAD of [M - 151- precursors derived from bis(acy1) glycerophosphocholines yielded carboxylate anions in a ratio of ca. 1:3 for the snl and sn2 substituents. (Interestingly, Murphy’s group has demonstrated (H14) that the [M - 151ion is derived from decomposition of a matrix/phospholipid adduct.) Huang et al. (H13) suggested, however, that this means of distinguishing the snl and sn2 groups was unreliable when the snl substituent was much larger than the sn2 substituent. These authors instead recommended differentiation based on the relative abundances of product ions derived by loss of the free acids from [M - 861- (phosphatidic acid anion) precursors; the product derived by loss of the sn2 acid was the more abundant in all the examples studied. Cole and Enke (H9)have suggested screening strategies for the analysis of several classes of phospholipids based on constant neutral loss and precursor ion scanning of positive ions. Kayganich and Murphy (H11) have cautioned, however, that the relative abundancesof different phospholipid components evident from such analyses may not be reliable if glycerophospholipids other than bis(acy1) species are included. There is an increasing realization that the development of atmospheric pressure ionization methods provides greatly improved facilities for the analysis of polar, low molecular weight compounds such as are important in xenobiotic metabolism studies. DiDonato and Gaskell (H15)reviewed the key advantages and discussed some examples. An extensive literature has yet to develop but some elegant applications have been described following the pioneering work of Henion and co-workers. (See ref H16 for recent examples from Henion’s laboratory.) Baillie and co-workers (HI 7), for example, have described qualitative and quantitative analyses of N-acetylcysteine and glutathione conjugates of valproic acid in the rat using HPLC coupled with ion spray (nebulizerassisted electrospray) tandem mass spectrometry. Baillieand Davis (HI 8) have provided a general perspective on the use of mass spectrometry for the characterization of glutathione conjugates, with particular emphasis on the application of LC/MS and tandem MS techniques. Fenselau’s group has also been active in the analysis of glutathione conjugates using tandem MS (H19, H20); in this instance, analyses have been performed using high-energy CAD on a four-sector instrument. Some differences between high-energy and low-energy (H18, H21) decompositions were observed, notably the prominence of ion types corresponding to charge retention on the intact glutathione moiety. Screening procedures based on constant neutral loss scanning or precursor ion scanning (H22, H23) are less readily performed using a four-sector instrument rather than a triple quadrupole or hybrid. Fenselau and Smith (H24) have reviewed the application of four-sector instruments to metabolism studies.
Tandem MS has been used extensively by Murphy and co-workersfor the characterization of eicosanoids (H25, H26); reviews have recently appeared (H27, H28). Schweer and Fischer (H29) described the CAD of [M - HF]- ions derived from pentafluorobenzyloxime derivatives of prostaglandins. Application to quantitative analysis was suggested but the same group elected to use methyl oxime, pentafluorobenzyl ester, trimethylsily ether derivatives for the quantification of prostaglandin E l and its major metabolites in human plasma (H30). Quantification during GC/negative ion tandem MS was by selected reaction monitoring of trimethylsilanol losses from [ M - pentafluorobenzyll- ions. Deuterium-labeled analogues served as internal standards. In general, however, applications of tandem MS to rigorous quantitative analysis remain few and far between.
ACKNOWLEDGMENT We acknowledge discussions and information from many colleagues too numerous to cite individually. A.L.B. acknowledges use of facilities and the warm and gracious hospitality shown during the preparation of the manuscript by Dr. Michael Waterfield and the members and staff of the Ludwig Institute, London. 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. R.K.B. thanks Jonathan Curtis, Mike Morris, Mike Quilliam, and Pierre Thibault for contributions and helpful criticism, and Ed Dyer for technical assistance with translating documents from Wordperfect into MacIntosh Word. Financial support was provided by NIH NCRR, Biomedical Research Technology Program Grant RR 01614 (to A.L.B.), NIEHS Superfund Grant ES 504705 (to A.L.B.), NIH Liver Center Grant AM27643 (to A.L.B.). NIH Grants GM 48267 and 45925 (to S.J.G.), and the University of Manchester Institute for Science and Technology. I .
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(820) (821) (622) (823) (B24) (825)
LITERATURE CITED A. OVERVIEW
(Al) (A2)
(A3) (A4) (A5) (A61 (A7) (A8)
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C. INNOVATIVE TECHNIQUES AND INSTRUMENTATION
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Q. CARBOHYDRATESAND GLYCOCONJUQATES (Gl) (G2) (03) (G4) (05) (G8)
(G7) (08) (G9) (G10)
(G11) (G12) (013) (G14) (G15) (018) (017) (G18) (019) (020) (G21) (G22) (G23) (024)
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