Anal. Chem. 1992, 64, 467 R-502 R (F8) Pomeroy, R. S.; Koiczynski, J. D.;Denton, M. B. Appi. Spectrosc. 1991. 45, 1111-1119. (F9) Webb, D. P.; Salin, E. D. Talents 1990, 37, 33-38. (FlO) Franqds, J.-P.; Janssens, F. Spectrochm. Acta 1990, 458, 177-188. (F11) Janssens, F.; Franpois, J.-P. Anal. Chem. 1991, 63, 320-331. (F12) Yang. J.; Plao, 2.; Zeng, X. Spectrochlm. Acta 1981. 468, 953-965. (F13) Yang, P.; Barnes, R. M. Spectrochh. Acta Rev. 1990, 13. 275-309. (F14) Boumns, P. J. W. M.; van Ham-Heijms, A. H. M. Spectrochim. Acta 1891, 468. 1545E-1568E. (F15) van der Mulien, J. A. M. Spectrmblm. Acta 1990, 458. 1-13. (F16) Burton, L. L.; Blades, M. W. Spectrmhim. Acta 1990, 458, 139-144.
(F17) Essottani, A,; Prouix, P.; Boulos, M. I.; Gieizes, A. J . Anal. At. Spectram. 1990, 5 , 543-547. (F18) Fannin. H. B. Appl. Spectrosc. 1990, 4 4 , 1143-1 146. (F19) Meadows, T. M.; Fannin, H. B. Spectrochim. Acta 1990, 458, 2 15-2 16. (F20) Curry, D. L.; Fannin. H. B. Appi. Spectrosc. 1991, 4 5 , 293-296. (F21) Sixta, V. At. Spectrosc. 1991, 12, 11-15. (F22) Bauer, 0.;WegscheMer, W.; Ortner, H. M. Spectrochim. Acta 1991, 468, 1185-1196. (F23) Thomas, R. J. At. Spectrosc. 1989, 10, 175-182. (F24) Watters, R. L. Jr. Spectrochim. Acta 1991, 468, 1593-1605.
Mass Spectrometry A. L.Burlingamel Department of Pharmaceutical Chemistry, T h e Mass Spectrometry Facility and the Liver Center, University of California, S a n Francisco, California 94143-0446
T.A. Baillie Department of Medicinal Chemistry, University of Washington, Seattle, Washington 98195
D.H.Russell Department of Chemistry, Texas A & M University, College Station, Texas 77843
A. OVERVIEW M w spectrometry has from its inception been an enabling
, revolutionizing the knowledge and understanding of every iscipline in its wake since the deflection of cathode rays by J. ( A l ) . Thomson ( A I ) . Mass s ectrometry’s most recent t r i m hs involve the new tools anfinstruments which have opened) up the prospect of studying macromolecular aspecta of cell biology and medicine. Now possible are studies embracing the discovery of how different t es of cells maintain their integrity, communicate with e a 2 other, and regulate the processes of life in whole organisms. The pathways underlying normal cellular function or those implicated in dysfunction, the many types of interactions of small molecules, both endo enous and exo enous, with cellular macromolecules of %oth beneficia! and toxicological importance-these are the frontier for mass spectrometric investigation. Pioneering work is underway on elucidation of macromolecular structure and biological function and delineation of the details of these interrelationships. The challe e is to follow the molecular fate of some loo00 proteins er c 2 t y p e , from the ribosome to their degradation in the bsosome-a life history of each protein, ita interconversions and transformations, interactions with other cellular constituents, and export or import into a cell, as well as its aid or subversion by components of microorganisms and viruses, or drugs and xenobiotics. Biological mass s ectrometry is both poised and destined to articipate fun8amentally in gaining knowledge of the morecular language which forms the common basis for understanding all living systems (A2).This assertion may appear at first sight to be hype, since mass spectrometrists have historically tended to oversell rematurely the importance of their methods in other discipenes (A3, A4). What is to say that this is not the case presently? In truth, in some respecta it will probably always be the case. However this danger can be minimized to a great extent by encouraging the practice and development of biolo ical mass spectrometry as an intellectual and experimend joint venture with biological and clinical scientists. Certainly, the most significant barrier to successful utilization of mass spectrometric developments in
techO1oY
biology and medicine is the mutual lack of experience in how to prepare, isolate and handle a precious sample in order to assure eventually successful mass spectrometric investigation (A5) (the so-called “front end” problem). This present weakness is exacerbated by the fact that the most important and biologically exciting problems tax the ragged edge of experience and existing technical ability, particularly in absolute sensitivity. On the one hand, the sheer numbers of such first rate opportunities far exceed the more easily tractable biological problems, but mention should be made of a few of such recent examples, including attempts at identification of low levels of 0-glycosylation in nuclear activation factors (A6),identification of only a very few of the lar e number of peptides detectable a t low levels in the higf ly complex mixtures presented by cell surface major histocompatability glycoproteins (A7,A8), and the determination of primary sequence which permitted the cloning and expression of the membrane bound sialyltransferase required for biosynthesis of the sialyl Lewis X antigen from a total of only 300 pmol of protein (A9). Both experience and optimized instrumentation are required to make headway on these types of problems. On the other hand, while significant advances in sensitivity have been made [multichannel array detectors (AlO, A l l ) , continuous flow LSIMS (A12),and matrix assisted laser desorption ion sources ( A l 3 ) for high-energy MS-MS are examples], even the best current mass spectrometric instrumentation is still seriously challenged by the most crucial biomedical problems within reach, including those mentioned above. These pressing problems provide the driving force and justification for continued technical innovation which will permit their solution with future generations of state-of-the-art research grade and commercial instrumentation. Consider one case in point: the disparity between the optimal performance characteristics of capillary zone electrophoresisand the operational sensitivity of virtually all presently available mass spectrometric instrumentation. The desirability of a marriage which would preserve the unprecidented resolution of separations with accurate mass spectral analysis is irrefutable. One further example is the possibility of taking
0003-2700/92/0364-467R~~Q,QQ/Q 0 1992 American Chemical Society
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A. L. Burlingame is Professor of Chemistry and Pharmaceutical Chemistry in the Department of Pharmaceutical Chemistry, University of California, San Francisco. He is also Director of the NIH-supported National Bio-organic, Biomedical Mass Spectrometry Facility and of the core mass spectrometry facility of the Liver Center 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 01 the structure of indole alkaloids. He imms diately joined the Department of Chemistry and Space Sciences Laboratory of the University of California, Berkeley, as Assistant Professor of Chemistry. He assumed his current responsiblllties in 1978. From 1964 to 1973, he was a member of several interdisciplinaryscientific 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 US. 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, high-sensitivity, high-resolution mass spectrometry, field ionization kinetics, and deuterium difference spectroscopy in NMR analysis. During 1970-1972 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 structural studies of modified proteins, receptors, and glycoconjugates of biomedical importance and the development of techniques and instrumentation to advance such studies of natural and recombinant biopolymers. 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. ~
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Thomas A. Balllie is Professor of Medicinal Chemistry at the University of Washington in Seattle, WA. He was educated at Glasgow University, where he earned his B.Sc. (Hons. chemistry, 1970) and Ph.D. (organic chemistry, 1973). He also received an M.Sc. (bis chemistry, 1975) from Chelsea College, London, and recently was awarded a D.Sc. (chemistry, 1992) from Glasgow University. His graduate work under Professor C. J. W. Brooks and postdoctoral studies as a Royal Society European Fellow under Professor J. Sjovall (Karolinska Institute) dealt with the application of GUMS and stable isotope tracer techniques to the analysis of steroids and their metabolltes in biological fluids. He new a lecturesnip at me Royal Postgraduate Medical School (University of London) from 1975 to 1978, following which he joined Professor A. L. Burlingame’s Mass Spectrometry Resource at the University of Califomia, San Francisco, where he was Assistant Professor in the Department of Pharmaceutical Chemistry from 1978 to 1981. He took up his present position at the University of Washington in 1981 and held a Senior InternationalFogarty Fellowship (1988-1989) during which time he conducted research on oxidative polymorphisms in drug metabolism at Huddinge University Hospital (Stockholm, Sweden). His current interests lie in the application of mass spectrometry and associated techniques to mechanistic studies in the fields of xenobiotic metabolism and chemical toxicoiogy.
Davld H. Russell is Professor of Chemistry at Texas A & M University. He received his B. Sc. from the University of ArkansasLittle Rock in 1974 and his Ph.D. in 1978 from the University of Nebraska. His graduate work, under the supervision of Professor M. L. Gross, involved studies of ion-molecule reactions and unimolecuiar dissociation reactions of gas-phase ions. As a Research Scientist at Oak RMge National Laboratory (1978-1980) he became interested in multisecetor tandem mass spectrometers and developed one of the ‘, , \ first EBE configuration tandem instruments. During this period he also became interested in photodissociation as a tandem mass spectrometry experiment and as a structural probe for gas-phase ions. Tandem mass spectrometry, gas-phase ion chemistry, and Fourier-transform ion cyclotron resonance (FTICR) spectrometry have been his major focal points of research since moving to Texas A & M University in the fail of 1980. His present research interests include tandem mass spectrometry, fundamental gas-phase ion chemistry, and FTICR spectrometry. However, in the past 2 years major emphasis has been given to expanding laser-based mass spectrometry experiments and the development of high-performance time-of-flight (TOF) methods. For example, a tandem magnetic sector (EB)/reflectron-TOF (R-TOF) instrument has been developed and a tandem R-TOFIR-TOF instrument is currently being built. The emphasis of the laser-TOF development is on new methods for analysis and structural characterization of large molecules at the femtomoie and subfemtomole sample levels. He is a member of the American Chemical Society, Society for Applied Spectroscopy, and the American Society for Mass Spectrometry. He is a coeditor for the Journal of Cluster Science and is an Editorial Board Member of Organic Mass Specfrometry. G
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advantage of Fourier transform mass spectrometry with new ionization techniques such as electrospray to measure small proteins, particularly those which mi ht be covalently modified, with higher resolving power anf better mass measurement accuracy (A14,A15). In addition, while there is still little practical utility at the present time, it appears that small, intact proteins can be collisionallyactivated in an electrospray ion source to form some product ions which can be assigned to sequences in known proteins used presently as models (A16). What are the chances that these so-called “front end” problems will be addressed and solved systematically,and that new generations of instrumentation with higher absolute sensitivities will be forthcoming? There is clearly the incentive in the biological and clinical community to exploit these revolutionary opportunities and new methodology in an integrated, cost-effective fashion. Now that the momentum is building to bring these exciting possibilities into the realm of biomedical reality, how will the mass spectrometry research community and the manufacturers be able to proceed? Unfortunately, there has been deepening trouble with regard to adequate financial support for both the development and the exploitation of biological mass spectrometry and other sciences dependent upon relatively expensive instrumentation, 468R 0 ANALYTICAL CHEMISTRY, VOL. 64, NO. 12, JUNE 15, 1992
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particularly over the last seven years (A17). More recently, this mtrend~ took major for the worse with a massive 73% m a~ ~ turn w cut in the Shared Instrument Program at the National Center for Research Resources (NCRR) at NIH (A17). This catastrophic truncation of Handler’s legacy (A18)is currently projected for the third straight year into FY 93 in the President’s budget for the NCRR Shared Instrumentation Program. At least in the United States, there is no alternate source of funding from which a group of academic biomedical scientists can even hope to obtain the capital necessary for purchase of critical, but relatively costly, shared equipment necessary to pursue these time-honored, cost-effective activities [“...first there must be instruments to share...” (A18)l. The rational notion of “interdisciplinary sharing” of rare expertise and state-of-the-art equipment is predicated upon the assured accelerated ace of discovery when “unique”new tools may be exploited. 8uch centers of excellence are designed to speed the study of the biology of human health and disease by interactive collaboration with a large number of experts in biology and medicine. The investments are long term by necessity and must be fostered by the keepers of our national trust. If not soon rectified, this national shortsightedness will conspire to preclude American biomedical scientists from participation in the timely realization of the undeniable, revolutionary insights always inherent in the pioneering practice of critical new technologies in biological and clinical investigation. The toll will be taken in terms of the loss of discoveringscientific information and the increased personnel costs required for the continued pursuit of laborious, inadequate alternative experimental strategies.
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B. SCOPE These reviews continue to be a testament to the irrefutable importance of sustained research and development efforts on new ion optical designs optimized to exploit the innovations in ionization methods. Such innovations benefit a broad spectrum of science at the atomic and molecular level, from physics to material science, astrochemistry, surface science, geochronology,...,to the life sciences and rational drug design for both the academy and biotechnology industry. Similarly, expertise in the optimization of instrumentation in any of these fields leading to many serendipitous breakthroughs requires a cadre of dedicated scientists and engineers functioning as a team for oftentimes their entire careers. These new methods do not always replace established techniques in mass spectrometry, as witnessed by the rapid identification of the novel amino acid associated with eosinophilia-myalgia syndrome responsible for 27 deaths and over 1500 cases as of August, 1990. Accurate mass measurement of a liquid SIMS molecular ion gave a molecular formula which was important
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in identification of this contaminating toxic byproduct as 1,l’-ethylidenebis[tryptophan] ( B l ) . A review such as this must be selective, as it has been in the past (B2). Every attempt has been made to illustrate the topics chosen with the most interesting problems and newer methods, but of course the detail and documentation which the reader may desire must be found by consulting the or literature cited. This work continues the continuity o the past and many important points made in previous reviews are still as relevant as they were a t the time of publication (B2). Again, we would recommend consultation of these for the reader to understand the chain of activity and progress over several years. While this review treats the protein and carbohydrate aspects of macromolecular systems preferentially, one must not lose sight of the continuing outstanding contributions being made in nucleic acid structural elucidation. This important topic has been summarized recently b McCloskey (B3),but a few additional contributions should ge emphasized. These include the posttranscriptional modification of transfer RNA two novel nucleosides of mRNA in thermophilic archaea (B4); cap 4 from trypanosomatids (B5);a rare study of mRNA mechanism of conversion of adenosine to inosine by doublestranded RNA unwindin modifying activity (B6);and one of the most extensive stuiies of the use of stable isotopes to understand the collision induced dissociation of heterocycles (B7). This topic raises the question as to why no one has yet come up with a competitive strategy for sequencing DNA based on some form of mass spectrometry. There is certainly interest in the subject as well as incentive, since the error rate in DNA sequences determined by existing technologies is in the order which is a potential source of grief (B8,B9). of 1in lo00 (B8), We apologize for leaving out many other important topics and issues at this juncture, but many of these have and will be treated in Mass Spectrometry Reviews (BlO). Mass Spectrometry Reviews (BlO)is in its 11th volume and has a new co-editor, Dr. Nico Nibbering of the University of Amsterdam, together with Dr. Maurice Bursey of the University of North Carolina. The Journal of the American Society of Mass Spectrometr is now in its successful third volume, with a backlog ( B l l c The working group on organic trace analysis of IUPAC has recently issued a report on mass spectromet and trace organic analysis (B12). The new important p e z index of the registry of mass spectral data has recently been published (B13) and described (B14). The comparative evaluation of mass spectral databases has been presented (B15). As usual,mass spectrometry is involved in a broad spectrum of activities in science, including synthesis of bis(Buckminsterfullerene)nickel cation in the gas phase (B16),its use in tandem with laser spectroscopy (B17),and in probing the mysteries of ancient Egypt (B18). Interestingly, these resin acids identified in a Roman period Egyptian mummy (B18, B19) are also found in aeolean dust and deep sea sediments (B20). The development of accelerator mass spectrometry was clear for the purpose of retrospective mapping of the actual neutron fluences in Nagasaki and Hiroshima but also romises to be a valuable technique in biomedical dosimetry 6 2 1 ) . It is able to measure radionuclides at orders of magnitude below that which is possible b scintillation countin (B21, B22). Hi h mass ions: how gigh is high? John %,nn reports po&(eth lene glycols) with molecular weights up to 5 million (B23). Jerhaps the measurement of the sizes of cellular organelles will be next. Mass spectrometry is routinely used for monitoring and controlling the gas levels in anaesthesia, but recently has been ap lied to the study of the pharmacokinetics of penicillin G diredy into the bloodstream of a live rat (B24). Reports of the 38th and 39th ASMS Conference on Mass Spectrometry and Allied To ics (B25, B26) have been published, and the 40th t h o he!t 45th Conferences will be held in Washington, D.C., San%rancisco, CA, Chicago, IL,Atlanta, GA, Anaheim, CA, and Kansas City, MO, respectively. An International Conference on Biological Mass Spectrometry will be held in Kyoto, Japan, the week of September 20,1992 (B27). The Montreux Conference on LC-MS will be held in Montreux, Switzerland, November 44,1992 (B28). The third conference in a series on Mass S ectrometry in the Health and Life Sciences will be held in &n Francisco in September
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1994 (B29),just after the 13th Intemational Mass Spectromet Conference, to be held in Budapest in August 1994 (B30). zeveral volumes of importance have appeared during this period, including Stable Isotopes in Pediatric Nutritional and Metabolic Research edited by Chapman et al. (B31);Liquid ChromatographylMass Spectrometry: Applications in &ricultural, Pharmaceutical and Environmental Chemistry, edited by Brown (B32);Volume 193 in the Methods in Enzymology series edited by McCloskey (B33);Mass Spectrometry of Biological Materials, edited by McEwen and Larsen (B34); a volume edited by Gross entitled Mass Spectrometry in the Biological Sciences: A Tutorial (B35); Biomedical Applications of Mass S ectrometry, co-edited by Suelter and Watson (B36);and a vofume on Continuous-Flow Fast Atom Bombardment Mass Spectrometry, edited by Caprioli (B37). Also of interest are: Mass Spectrometry of Peptides, D. M. Desiderio, Editor (B38);Mass Spectrometry in Pharmaceutical Research Current Boundaries and Future Frontiers co-edited by Garland and Vanden Heuvel (B39); a book of the proceedings of the Second International Symposium on Mass Spedrome in the Health and Life Sciences, entitled Biological Mass pectrometry, Burlingame and McCloskey, Editors (B40);and Volumes I1 and I11 in the Techniques in Protein Chemistry series, edited by Villafranca and Angeletti, respectively (B41, B42). A few noteworthy conference proceedings have appeared in journals, such as the Proceedings of the 7th International Symposium on Liquid Chromatography-Mass Spectrometry, Supercritical Fluid Chromatography-Mass Spectrometry, Capillary Electrophoresis-Mass Spectrometry, and Tandem Mass Spectrometry, Montreux, Switzerland, published as a volume of Journal of Chromtography (B43);the Proceedings of the Second International Symposium on Applied Mass Spectrometry in the Health Sciences, Barcelona, Spain, also appearing in Journal of Chromatography (B44);and the Proceedings of the 12th International Mass Spectrometry Conference,Amsterdam, The Netherlands, which will be published as Advances in Mass Spectrometry, Vol. 12, as well as two special volumes of International Journal of Mass Spectrometry and Ion Processes (B45, B46). Two periodicals of interest are GC MS Update: Part A (Environmental)and Part B (Biome ical, Clinical, Drugs), Down and Halket, Editors, and LCIMS Update, also edited by Halket and Down. A selection of review articles primarily of interest in biological applications of mass spectrometry include one by Hill on “The Widening Horizons of Bioanalytical Mass ” (B49);an article by Shackleton entitled “Stable Isotopes Spectrome?$ in teroid Research” (B50);an article by Stroh and Rinehart, “Liquid Chromatography/Fast Atom Bombardment Mass Spectrometry” (B51);“Analym of Protein Structure with Mass Spectrometry”by Fenselau (B52);“New Developments in Biochemical Mass Spectrometry: Electrospray Ionization” by Smith et al. (B53); an article on “Integration of Mass Spectrometry in Analytical Biotechnology”by Carr et al. (B54) and another b Carr on “Recent Advances in the Analysis of Peptides a n 2 Proteins by Mass Spectrometry” (B55); “Development of the Electros ray Ionization Technique” by Hamdan and Curcurato (B567; and “The Recent Evolution of the Quadrupole Ion Trap Mass Spectrometer-An Overview” by Todd and Penman (B57).
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C. INNOVATIVE TECHNIQUES AND INSTRUMENTATION The experimental methods a t the forefront of analytical mass spectrometry are dramatically different from standard mass spectrometry ionization/excitation techni ues, e+, electron impact, chemical ionization, collision-inJuced dissociation, etc. Consequently, new instruments and new versions of old instruments have appeared in the last several years. For example, the applications of time-of-flight (TOF) mass spectrometry are once again growing very rapid1 , and mass resolution in excess of 20 OOO has been reporteJ(C1). Quadrupole ion traps are quickly finding their place in analytical mass spectrometry. Electrospray ionization (ESI), originally introduced by Dole more than 20 years ago, is having profound influence on biological mass spectrometry. Techniques such as 262Cf-plasmadesorption, multiphoton ionization, laser desorption, and electrospray ionization are powerful analytical methods, but are more readily adaptable to nonANALYTICAL CHEMISTRY, VOL. 64, NO. 12, JUNE 15, 1992
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conventional mass spectrometers or require specialized interfaces. The ionization methods that have expanded the scope of biological mass spectrometry have also catalyzed development of time-of-flight (TOF), quadrupole iontraps (QIT) (C2), Fourier-transform ion cyclotron resonance (FTICR) mass spectrometers (C3),and a modern detection system (microchannel plate arra detector) for focal plane mass spectrometers (C4-C6). In adhition, the newer analytical mass spectrometers are benefitting from many years of instrument development in the area of chemical physics, e.g., utilization of nozzle-molecular beam sources and skimmers for achieving the high vacuum requirements of the analyzer (C7, CS). Laser systems have become standard inventory for most and the introduction of mass spectrometry laboratories (C9), pulsed-UV-matrix-assisted laser desor tion ionization will certainly influence this trend (ClO).Jowever, photodissociation has been around since 1980, and lasers have been used as ionization sources in mass spectrometry since 1973. Although much of the literature on laser ionization (especially for organics) deals with multiphoton ionization schemes, Becker has recently reported on single-photon ionization of organics by 10.5 eV photons (frequency tripling of 354.7 nm (Nd:YAG) to yield 118.2 nm) ( C l l ) ,and Johnston has used this same method to stud photoionization of neutral fragments formed by photo2ssociation of neutral molecules (C12-C14). These methods are applicable to a range of different studies, e.g., post-ionization of desorbed neutral molecules, direct ionization sources for organic molecules (C15), etc. Such methods might prove of greater general utility because the ionization is not restricted by optical selection rules as is multiphoton ionization. Time-of-flight (TOF) mass spectrometry has undergone dramatic changes since Macfarlane introduced 252Cfplasma desorption in the early 1970's (C16). Although TOFMS is generally regarded as a low-resolution, low-sensitivity instrumental method, the changes in instrument design and electronics afford both high mass resolution and hi h sensitivity (C17). The "renaissance of time-of-fliggt mass spectrometry" is well documented in a recent review by Price (CIS)and in a previous review published in 1984 (C19). Several apers on details of TOF instrument designs have appearefthat focus on resolution and sensitivity issues (C20, C21). On examining the literature on TOF mass spectrometry one is impressed with the diversity of areas of development. The development of time-array detection (TAD) by the Michigan State University group has opened new areas of high speed hy henated mass spectrometry. For example, TAD GCT8FMS provides significant improvements over scanning mass spectrometers; up to 20 mass spectra per second (faster data acquisition rates are possible) have been generated to accurately reconstruct the chromatographic profile (C22). TAD TOF methods have also been used for LC/MS (C23), and AD/TOF combined with CZE is in the developmental stages (C24). The high speed TAD data acquisition rate permits optimum chromatographic resolution by the CZE. There has been considerable recent progress in tandem mass spectrometry TOF experiments. Because this is a developing area, much of the emphasis has been on answering fundamental questions, such as how kinetic energy released upon dissociation effects peak shapes (CW),but signifhnt advances have been made in developin high-resolutionlhigh-sensitivity tandem MS experiments (826-C28). Tandem TOF mass spectrometry is an emerging area that will likely have significant impact on bioanalytical applications, especially in combination with laser ionization [including matrix-assisted laser desorption ionization (MALDI)] and chromatographic separation methods. The quadru ole ion trap mass spectrometer has undergone significant anfrapid developments in the past 2 years. The developments are described in a number of topical reviews and overview papers (C29433). Many aspeds of the ion trap are desirable in terms of resent trends in analytical mass spectrometry. For exam ye, chromatography-MS methods are not directly compatibg with the vacuum re uirements of mass spectrometry, especially FTICR thus, elaorate interfaces must be developed. Methods such as electrospray ionization, which are even more restrictive in terms of the source/analyzer interface, are becoming increasingly important
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to biolo ical mass spectrometry; thus, instruments more amenabk to the vacuum requirements are beneficial. Matrix-assisted laser desorption ionization, another major innovation in biological mass spectrometry, yields ions that have appreciable kinetic energies that adversely affect the mass resolution of time-of-flight measurements and ion trap @/ion detection for FTICR (C34). The bath gas requireffor o eration of the ion trap collisionally relaxes the translationdy excited MALD formed ions, minimizing any potential adverse effects of the ions' kinetic energy. In early experiments, the mass range of the ion trap was restrictive in terms of a biological mass spectrometer; however, recent results illustrate that ions of 10-30 kDa can be analyzed by this method (C35, C36). The successful adaptation of electrospray ionization adds a new dimension to high mass analysis (C2, C37, C38). In addition, major improvements in mass resolution have been demonstrated (C39, C40), as well as an endless variety of tandem mass spectrometry experiments ( C 4 1 4 4 9 ) . The impressive developments in the ion trap and adaptation of an assortment of essential accessories place this instrument on a level playing field with all other instrument technologies. The following questions, however, remain to be answered (i) can the instrument technology be exported from the specialist laboratory to the analytical laboratory and retain the demonstrated level of performance; (ii) are the slow scan rates required for high-resolution mass measurements prohibitive to the development of an application instrument, especially chromatography-MS (iii) having demonstrated an acceptable level of mass resolution, can mass measurement precision be achieved at a level comparable to current state-of-the-art;and, finally, (iv) what is to be the cost of such an instrument? Fourier-transform ion cyclotron resonance (FTICR) mass spectrometry is still in the development stage, and the major focus at this time is in the area of detecting high mass ions. Grosshans and Marshall have published a new version of the general theor of excitation for FTICR ((250). Two points should be male concerning this paper: (i) great detail is given to the mathematical description of the excitation process, and this alone is a significant contribution to the field; (ii) the model assumes "negligible initial cyclotron radius" for the ion prior to application of the excitation rf field. The latter point is oftentimes overlooked when discussing detection of high mass ions by FTICR, i.e., the radius for thermal energy ion increases as the m / z of the ion increases and the ion radius increases as the initial kinetic energy of the ion increases. Because ionization methods such as MALDI and other desorption ionization methods form ions with appreciable kinetic energies, the fundamental assumption of the ICR theory (as initially derived and as restated by Grosshans and Marshall) cannot be overlooked! Initial kinetic energy of the ion effects both the translation energy distribution of the ions followin excitation as well as the phase coherence of the accelerate3 ion ensemble (C51). Similar facts should also be considered when ions are formed in an external ion source and injected into the ICR cell. The kinetic energies of the ions will influence ion trapping and detection, and z-axis motion will cause time-dependent variations in the measured ion abundance (C52). The "infinity cell" described by Caravatti and Allemann has advantages for detection of high-mass ions and for eliminating effects of z-axis energy (C53). These points have been made previously by both Marshall (C54) and Russell (C55). In spite of the apparent difficulties that hi h-mass FTICR must overcome to expand as a routine met od, impressive progress has been reported. Comisarow and co-workers have continued with the development of laser desorption ionization FTICR for the analysis of oligosaccharides, extending this work to systems containing neuraminic acid and pyruvic acid (C56). Brenna and Creasy have reported on the analysis of neutral olymers (C57). Hettich and Buchanan have continued tge development of MALDI with FTICR (C58),and McLafferty has successfully demonstrated electrospra FTlCR (C3). A recent paper from McLafferty's grou und7erscores the need for continued develo ment in the un1erstanding of ion trapping/detection by F d C R (C59). The authors report increased signal-to-noise ratios for multiple remeasurement of ions formed by 252Cf-PDMSand laser desorption, Le., ions are formed over some time period and the same ensemble of ions are detected n times. This ion detection method gives an enhancement in the S/N ratio of 4X for 25 measurements.
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It is unlikely that the physical processes occurring within the ion cell are as simple as depicted by the authors, but it is clear that additional work should be performed, Very recently Solouki and Russell, and Wilkins and co-workers have reorted excellent results for MALDI of high mass peptides. or example, Solouki and Russell reported a spectrum of bovine insulin with excellent signal-to-noise (>100:1)(C34), and Wilkins' group has reported spectra for peptides/proteins above 30 kDa molecular weight (C60). Structural FTICR also continues to advance at a reasonable paca. Both W U (C61)and McLafferty (C62)have reported methods for performing surfaceinduced dissociation. Because each of these experiments involve manipulation of the ions in such a manner as to affect ion trappingldetection, the multiple collision off-resonance CID experiment described by Jacobson may ve greater analytical utility (C63).Other approaches to structural FTICR include 193-nm photodissociation (C64, C65), dissociative chemical ionization of laser and MPI of laser desorbed neutrals desorbed neutrals (C66), (C67). Laser desorption ionization has been viewed by many as a potentially powerful analytical method for biological mass spectrometry, but the major developments in this area occurred following introduction of "matrix-assisted" laser desorption ionization (MALDI). MALDI differs from other laser desorption ionization (LDI) methods in that a W laser is used to excite an electronic transition of the matrix molecule. Typically, matrices such as nicotinic acid or 3,5-dimethoxy4-h droxycinnamic acid (sinapic or sinapinnic acid) are used as &e matrix, but other substituted aromatic compounds have been used. For example, molecules such as thymine, 31alcohol, 4-nitroaniline, 2,4-dinitroaniline, etc., have g : k T f o r MALDI. The physics and chemistry of MALDI are not fully understood (C68).For instance, it is not clear whether the matrix most strong1 affects the desorption or the ionization process or both! ?hat is, the absorptivity of the matrix causes more energy to be deposited into the sample, thereby resulting in the desorption of more material (C69, C70),and it is possible that the matrix plays a key role in the production of ionic material (C71).Should we refer to MALDI as matrix-assisted desorption or matrix-assisted ionization? The laser excitation produces a plume of desorbed material that behaves as a free 'et expansion (C72,C73),and collision within the plume resdts in cooling as well as proton-transfer reactions. A number of non-mass spectrometric studies have characterized the plume, m terms of spatial and temporal distributions of large molecules (C74),the survival of large complex single-stranded DNA (C75),and fluorometric detection of laser desorbed species (C76).The non-mass spectrometric studies provide a view of the desorption rocess that cannot be seen by only detecting the ionic pro8ucts. We know that it is essential that the matrix absorb the incident radiation from the laser. Studies of postionization MALD formed neutrals show that pro er selection of the matrix influences the density of desorbefneutrals (C77),and it was suggested that the matrix also promotes ionization of the analyte molecule (C78).Recent studies from Russell's laborato correlate the efficiency of several matrices to the enhancer acidity of the electronically excited state of the matrix molecules (C79).The observation that sinapic acid methyl ester is equally as good a matrix as sinapic acid is consistent with this hypothesis, but clearly more work in this area is required. The ratio of matrix and analyte influences both the abundance of matrix and analyte ions (C80).The photochemistry( photophysics of organic molecules is a thoroughly studied area (not to imply a high level of understanding), thus we should be able to design highly efficient systems for MALDI. In addition, we should be able to utilize such studies to gain further understanding of condensed phase photochemistry/photophysics. MALDI has clearly brought new energy and enthusiasm to biological mass spectrometry of large molecules, but, in our excitement to detect larger and larger molecules, we should not lose sight of the fact that we have a new method to study the mechanism(s) of desorption ionization. Because MALDI is initiated by excitation of a specific electronic state of the matrix molecules, we have controls that are not operative in other desor tion ionization experiments. For example, Beavis and CRait (C81)and S en ler and Cotter (C82)measured the kinetic energies of dAL%I formed ions and obtained vastly different answers.
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At this juncture, the actual kinetic energies of the MALDI formed ions are less important than understanding the factors that influence the ion kinetic energies. For instance, are the ion kinetic energies dependent upon the matrix, the matrixto-analyte ratio, the laser fluence, etc.? For a matrix such as sinapic acid that has optical transition in the 350- and 250-nm region, do the kinetic energies of the MALDI formed ions depend u on the optical transition that is "pumped" by the incident k e r ? Two of the pioneering laboratories of MALDI have repared a review that presents an overview of applications ani leading references on matrices and sample preparation (CIO).Other review-type papers have also appeared on MALDI (C83)that illustrate the range of uses of this method. For example, Beavis and Chait have developed methods for determining the molecular weights (to 0.01% up to 40 kDa.) (C84)of Spengler et al. have approteins in complex mixtures (C85); plied the method to determination of underivatized oligoand the method has been used with deoxynucleotides (C86); IR (C87,C88)or low-power (1.2 mJ) pulsed N2 lasers (1289). Laser desorption ionization (LDI) of frozen aqueous solutions of biomolecules has produced intriguing results both in terms of the ionization process and post-ionization of neutral fragments. For example, Williams showed that sodium salts of oligodeoxyribonucleic acids (DNA) (MW 18.5 kDa) and a protein, lysozyme (MW 14205), can be ionized as intact molecules by irradiating the frozen aqueous solution with visible light (C90, C91). If the irradiating laser is tuned to either 589 nm (Na D-line) or 578 nm (Cu atomic transition; a Cu sample support was used), then the yield for the intact ion of the sample increased by approximately 1 order of magnitude. It would be interesting to consider the parallels between this experiment, MALDI usin or anic matrices, and LDI from glycerol matrices and "fmely divi6ied metal particles" (C92),assuming there are any. Although such studies may lead to better, more efficient applications of MALDI methods, it may be equally important to unravel fundamental processes involved in desorption/ionization. Electrapray ionization (ESI)has influenced biological mass spectrometry in a manner similar to MALDI, although ESI has had possibly an even greater impact. Specific applications of ESI are discussed in a separate section of this review, but a few noteworthy applications that range beyond those of protein chemistry include the analysis of ionic transition metal complexes (C93),a traditionally difficult sample for mass Spectrometry, and porphyrins (C2).In addition, ESI has been used to detect hemeglobin complex in native myoglobin (C94) and an imine intermediate on dehydroquinase (C95).The range of protein chemistry problems that can be addressed by ESI illustrates the impact that this method is having on biological mass spectrometry, and fundamental studies of the ionization process are occuring parallel with applications. Kebarle and co-workers have compared ESI and ion spray, in terms of the mechanism($ of ion formation (C96, C97). The dependence of ion yield on the presence of electrolyte suggesta the charging is electrophoretic. A later study suggests that the ion yield is weak1 dependent upon the nature of the electrolyte, because H81 and NaCl give slightly different ion yields (C98). Rapid progress is also being made in direct introduction of liquid samples. Methods such as thermospray and electrospray sample introduction are having profound effects on biological mass spectrometry (CSS). New methods may extend the range of biological mass spectrometry. For example, aerosol generators are effective nebulizers and offer unique advantages for introduction of nonvolatile and or thermally labile sam les (C7,C8). Although the reporte studies have relied on E# for ion formation, other ionization methods (ClOO) and possibly laser ionization methods offer a higher degree of selectivit in terms of the ionization process, greater sensitivity, a n J a d a tability to a greater ran e of instruments. The use of mem ranes for selective introluction of volatile or anic compounds has matured. To apply this method to pofar, ionic compounds requires the synthesis of new polymers that are compatible with the sample (C105,Cl06).
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D. TANDEM MASS SPECTROMETRY Tandem mass spectrometry has evolved as a standard method for structural mass spectrometry. This technique is an accepted method for amino acid sequencing of peptides ANALYTICAL CHEMISTRY, VOL. 64, NO. 12, JUNE 15, 1992
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(see Section E) and other classes of biomolecules, but the real power of tandem mass spectrometry is that structural studies can be performed on samples that are less than 100% pure. In fact, tandem mass spectrometry was developed as a mixture analysis method. The area of hi hly s ecific dissociation reactions of metal ion-biomolecufe ad&cts would not be analytically useful without tandem mass spectrometry (Dl, 02). Altho h the em hasis of metal ion-biomolecule adduct studies hasTeen one!t site of attachEent of the metal ion (DS08), such studies have significance for structural analyis Dl0). Issues related to the site of complex biomolecules (D9, of attachment of the metal ion to the biomolecule are often based on ar uments on the solution state structure of the organomedic complexes. Implicit in these arguments is the assumption that the metal ion affinity of the biomolecule, s ecifically the functional groups of the biomolecule to which e!t metal ion is attached, are independent of the internal energy of the ion. This assumption is certainly subject to exceptions, assuming that it has any validity at all. In fact, it is this very assumption that leads to differing views on the site of attachment of alkali metal ions between Russell and Gross (and later Adams, Leary, and Gross). It seems highly unlikely that the site of binding of a metal ion to a molecule having sufficient energy to dissociate is adequately modeled by a ground-state structure of a solution- or solid-state molecule. In addition, the gas-phase dissociation reactions of the ion are unaffected by the structure of the solvent shell and ion-ion and/or ion-dipole interactions. Thus, such studies are important to both developing a clear understan of the dissociation reactions of complex molecules an structure effects imposed by solvation and solution/solid-state interactions. Furthermore, tandem mass spectrometry, with the ability to mass-select specific ions, is uniquely suited to studies of the chemistry of such systems. The issue that must still be resolved relates to methods of probing the mass selected ion. Structural tandem mass s ectrometry of com lex biomolecules utilizes collision-inAced dissociation ({ID) to activate the ion under investigation to energies above the dissociation threshold. The CID experiment can be performed by using tri le quadrupole instrumenta, ion traps [rf quadrupole trap an Fourier-transform ion cyclotron resonance (FTICR)], and magnetic sector instruments. The available collision ener ‘ea(laboratory frame) range from a few eV to several keV. flternative excitation methods, e.g., surfaced-induced dissociation (D11-D13), electron-induced dissociation (D14), and photodissociation, are active research areas but the major analytical method is CID. Variants of CID now include neutralization-reionization (D15) and neutralization-chemical reionization (D16). The mechanism by which translational energy of the incident ion is converted to internal ener (the target gas is t ically at thermal energy) has been stuxed in considerable Ztail. Collisional activation, the process of converting translational ener into internal energy, and subsequent reactions of the cofiionally activated ion must be considered separately. Durup’s original treatment of collisional activation/collision-induceddissociation (CA/CID) considered four limitin cases: (i) electronic excitation (a vertical excitation process$, (ii) elastic scattering off a single atom of the molecules, (iii) energy transfer in a long-lived ion-molecule complex, and (iv) excitation to a repulsive electronic state and dissociation. The commonly held view is that CA/CID of large molecules at low collision ene ’esfavors vibrational excitation of the ion, whereas at keV czision energies both electronic and vibrational excitation occurs. It should come as no surrise that recent studies contradict the folklore! There will e more discussion of this issue in the following sections. have reviewed the current status Bordas-Nagy and Je of the U n d e r s t a n d i n g T I D of di- and polyatomic ions drawing on the fundamentals of CID of diatomics to understand the case for polyatomic ions (D17). This review rovides a good overview of the excitation mechanism antenergy deposition and the effects of the collision gas. Cooks has examined the angular dependence of internal energy distributions as a means to estimate the contributions of vibration and electronic excitation mechanisms in keV energy CID (D18). The authors suggest that keV energy CA/CID involves primarily vibrational excitation, but a low-energy component may be due to electronic excitation. Naylor and Lamb have studied the effect of different target gases on low-energy CID
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of peptides and observed that the yield of positive ions is maximized when acidic gases are used (D19). This result is rationalized on the basis of a proton-transfer reaction from the target as to the amide nitrogen of the peptide bond that promotes fissociation, a mechanism similar that proposed by Fenselau (020) and Bursey (021). Holmes has published an interesting aper recently on radiative decay of collisionally activatecf ions, e.g., decay processes of ions that must involve electronic excitation at keV energies (022). Such measurements can be used to distinguish between isomeric ion structures, a point that is illustrated in the paper, and this experiment may prove 2 h l y useful in examining the fundamentals of keV energy CAf ID. In fact, one might question why mass spectroscopists and ion chemists have not utilized radiative decay studies to examine other ionization/excitation processes. It is highly probable that radiative decay processes preempt many processes important to analytical and physical mass spectrometry, e.g., excitation/dissociation of [M + Na]+ type ions, desorption ionization of samples containing residual sodium or other alkali metal ion salts, desorption ionization, especially MALDI of molecules that contain fluorophores, and sample containing impurities that promote intersystem crossing to the triplet manifold. Another interesting experiment that may have ramifications for structural mass spectrometry and possibly even tandem mass spectrometry involves the use of 118-nm photons for ionizin (via single-photon processes) neutrals formed b laser abfation (023). The results of this study suggest t at a laser ionization counterpart to neutralization/re-ionization may be feasible and even of analytical utility. Such an experiment would be similar to experiments involving multiphoton ionization/laser photodissociation and/or laser desorption/laser photodissociation (024, D25). The objective of performing CA/CID is to produce a lar e number of fragment ions that can be assigned to speciic structural features of the neutral molecule. Because the possible dissociation pathways available to the ion span a range of internal ener ‘es,it is desirable to deposit a range of internal ener ies into t f e ion. In addition, because the most structural7y informative ions arise by simple cleavage reactions, .,high energy reaction channels, it is necessar to deposit a arge amount of internal energy into the ion. dternatively, deposition of a smaller increment of energy per collision under multiple collision conditions is a practical means for performin CA/CID. In practice, there are relatively few published 8ID spectra that were obtained under “single collision” conditions. [Recent work by Futrell’s group suggests that the design of collision cells used for magnetic sector instruments discriminates ainst (due to scattering) product ions formed by multiple c3lisions (0261.1 The most commonly used method for estimat’ the collision gas pressure in the collision cell is by observing%e attenuation of the incident ion signal, and the CA/CID conditions are usually reported as ya collision cell pressure corresponding to X% attenuation of the ion signal”. Unfortunately, beam attenuation is not a reliable method for estimating the pressure within the collision cell. If X is greater than 5-10% the number of incident ions undergoing “single collisions” is quite small, less than 30%. At beam attenuations of 25-50%, commonly used conditions for CID experiments, most of the fragment ions are formed by 3-5 collisions and a significant number of ions undergo as many as 5-8 collisions. It is relatively simple and highly informative to estimate the probabilitiesfor multiple collisions for a given beam attenuation. For example, at 25% attenuation the ratio of one/two/three collisions is 1/0.2/0.1; at 50% attenuation the ratio for 1-6 collisions is 1/0.5/0.24/0.12/ 0.08 0.07, and at 75% attenuation the ratio (1-8 collisions) is 1/03 0.7/0.5/0.4/0.3/0.2/0.15. In terms of the analytical CA/CI experiment the average number of collisions operative under a given set of experimental conditions is not all that important (027). The abundance of low mlz fragment ions may increase as a result of CID of fragment ions. In fact, the preferred beam attenuation may be 5&75% where the ratio of numbers of collisions is less sensitive to the collision cell pressure. Conversely, in studies of the fundamentals of CA/CID, specifically the energy transferred in the CA step, single collision conditions are required and the data is leas if acquired under conditions of multiple collisions. met od to discriminate against product ions arising by small impact parameter collisions and/or multiple collisions
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has been described by Boyd et al. (028).] For fundamental studies the ion beam-neutral beam method described by Futrell is the preferred method (029). In recent years there has been considerable controversy concerning the ener loss measurements accompanying CA of large molecules. gerrick pioneered this area and showed that energy loss accompanying CA increases as the mass of the incident ion increases. On the basis of ener loss measurements and previous studies on CA CID oflarge ionic systems, Derrick proposed a theory for CI6. The major points of the theory are (i) the average energy loss, (E),increases linearly with the energy of the ion, E; (ii) the aver e energy transferred to the ion, (Q), increases linearly with %e energy of the ion; (iii) the average energy transferred to the ion is directly proportional to the average energy loss; (iv) there is an approximate inverse relationshipbetween ( Q ) and the mass of the incident ion, mi; (v) the efficiency of transferring center-of-mass collision energy, E,, to (Q) increases with the mass of the neutral gas atom, ma, and the mass of the atom or functional group of mi are nearly equal; (vi) to a first approximation, increasing ma leads to a larger ( 8 ) ;and (vii) scattering increases when m, increases and when mi decreases. The CA/CID model, as presented, is a reasonable generalization of the available experimental results. Because the loss measurements performed by Derrick and also by g80.28) are not rformed under single collision conditions (typically 25-75% g a m attenuation), it is difficult to rigorously interpret these experimental results. In our own work, where we made ever attempt to work under single-collision conditions (10-20% Lam attenuation), we observed that the energy loss decreased as ma increased and the upper limit for the energy loss roughl correlated with the ionization energy of the target gas (0305: Consequently, energy losses greater than the ionization energy of m, can only occur by multiple collisions. The proposed charge transfer reactions have been criticized by both Boyd and Derrick, but the detection of product ions of endothermic charge transfer reactions by FTICR (031) as well as the detection of neutral species formed in collisional activation experiments (032) solidify the proposed char e transfer reaction. In addition, the first step in any neutrdzation-reionization experiment must involve an endothermic charge transfer reaction, and such reactions occur with all collision gases (015,033)! Boyd’s group made a strong effort to improve measurements of the energy loss accompanying CA/CID by using an electrically “floated” collision cell. One advantage of this approach is that dieaociation reactions occurring within the collision cell can be separated from dissociation reactions occurring outside . argues that “the corresponding the collision cell (034)Boyd values of AE (energy loss) should then be independent of collision gas presaue over a wide range”. Again, however, this statement assumes that all the chemistry occurs by single collision, and such an assumption is valid only for low percent beam attenuation but not under the 50% conditions used in this study. Another point worthy of more detailed study is the energy loss values for atomic target gases and polyatomic (diatomic) target ases. In our original study we noted that the energy loss v8ue for N2 did not correlate with the values expected on the basis of atomic gases. In subsequent studies we obCO, and COz did not yield served that diatomics such as 02, expeded energy loss values. In Boyd‘s studies he too observed that H2, D2, and N2 ave energy loss values considerably greater than He (for If2 and D2)and approximately that for Ar (for N 1. Differences may be due to different collision dynamics !or diatomics versus atomics. Greater collision cross sections for diatomics may give rise to different numbers of collisions for a given beam attenuation, and energy disposal may yield vibrationally excited target gas atoms. In all the studies reported to date on the energy loss process it was assumed that the inital internal energy of the incident ion has no net effect. This is probably a bad assumption in all cases and is especially bad in cases where the ions are formed with a broad range of internal energies (035,036), e.g., by ion-molecule reactions, particle-induced desorption ionization, and laser desorption ionization. Very recent studies in our laboratory showed that energy loss accompanyin CA/CID of glycerol cluster ions, e. [Mn + HI+, mode! peptide ions, and even-electron ions k m e d by EI, are quite s m d at low collision gas presaures (&15% beam attenuation),
but as the pressure is increased the energy loss increases linearly. These results would suggest that at low beam attenuation the “hot” ions are activated and dissociate; an argument consistent with Cook’s view on the internal energydependent collision cross sections (036).After the beam is depleted of “hot” ions the ener loss value increases as the diatomic gases the energy number of collisions increases. loss value increases approximately linearly and finally reaches a constant level. Futrell has published an interesting series of papers on the dynamics of collisional activation of small molecules that are significant for studies of CA/CID of lar e molecules. An especially interesting study was that of H8*+(propane; a model QET molecular ion) and C3H60’+ acetone; a model non-QET molecular ion) (037),but the reader is also directed to a paper on nitromethane which contains interesting results and an excellent overview on the fundamentals of CA and ion . latter paper proposes two mechanisms chemistry (035)The for formation of NO+ and NO2+and suggests that small impact parameter collisions induce isomerization and dissociation, that low-energy collisions produce electronically excited nitromethane ions occupying the sixth ionization band of the photoelectron spectrum, and that dissociation proceeds directly from the excited state. The C2H5+ions from C3H!*+ are described in terms of vibration excitation and redistribution of energy prior to dissociation. On the other hand, the CH3CO+ ions from acetone have been suggested to involve electronically excited acetone ions. The different behavior for the propane and acetone molecular ions is attributed to the “very different electronic band structures for the two ions”. Long-lived electronically excited C3&O’+ ions, the A state (OB), undergo collisional deexcitation and then dissociate to form the acetyl ion and methyl radical (039). More recent studies show that ground state C H O*+ions are collisionally activated (apto form the A state and dissociation proximately 5 e\ occurs directly from the excited state (040). Although there is considerable interest in photodissociation as a method for ion activation in tandem mass spectrometry, the numbers of studies on complex molecules or even model compounds of biological molecules are very limited. The advantage of photodissociation is that the amount of energy deposited into the ion is defined by the energy of the photon; issues of average energy uptake and what fraction of the energy loss is partitioned as internal energy of the ion do not complicate the issue. The most serious disadvantage of the method is that the yield of product ions is low if single- hoton absorption is maintained, and sample multiphoton &sociation prrequire a high degree of variability of the laser power density. Several laboratories have published photofragment ion spectra of complex molecules, but these spectra are composite metastable ion, CID, and photofragment ion spectra. The reason for this is the low efficiency for photodissociation and the background ion of large ionic systems (10-2-10-4) counts that arise by metastable ion and CID processes may be as much as a few percent relative abundance of the “main ion” beam. In order to obtain a true photofragment ion spectrum it is necessary to substract the background metastable/CID s ectrum. Substraction of the background spectrum can e! done by modulation of the laser source and synchronously detecting the ion signal (e.g., lock-in amplifier) (041) or by ion counting methods (042). Another problem that frequently complicates the acquisition of the photofragment ion spectrum is the increased neutral background that arises from the laser beam striking metal components within the vacuum system. This yields a photofragment ion spectrum that is very similar to the CID spectrum because the fragment ions are formed by collision with laser evaporated/ablated neutral species. Having sounded a precautionary note, it is interesting to examine recent published examples of photodissociation studies; a more detailed review of photodissociation/structural mass spectrometry is in press (043). Martin et al. examined the 193-nmphotodissociation of angiotensin 111and compared the data with the sequence ions observed by CID (044).The photofragment ion and CID spectra are very similar, a result that suggests the internal energies of the photoexcited and collisionally activated ions may be very similar. The same research group compared the photodissociation of peptide ions at 193 and 350 nm and found that photodissociation at 350
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nm re uired derivatization with a chromophore (dinitrophenyg ( 0 4 5 ) as re orted previously by Tecklenburg et al. ( 0 4 6 ) . The most agundant photofragment ions arise from fragmentation reactions adjacent to the derivatized residue. The more detailed studies of Tecklenburg suggest that the type of photofragment ions formed is highly dependent upon the ion lifetimes sampled and the excitation energy of the photon. Amster and co-workers (047, 048)compared the photofragment ion (193 nm) and CID spectra of several small peptides and, in contrast to Biemann’s studies, found that the high-mass fra ment ions tend to dominate the CID spectra and low-mass fragment ions dominate the photofragment ion spectra. Wilkins and co-workers described the combined use of hotodissociation and surface-induced dissociation (SID) for bS/MS/MS studies ( 0 4 9 ) . The authors report that combining photodissociation with SID increases the fragment ion yield, but the authors do not establish whether the increased ion yield is due to an increased photon absorption crow section for internally excited ions (e.g., following surface-induced excitation) or excitation of ions to higher avera ed energies as a result of surface-collision/photoexcitation. b h e authors do report that photoexcitation impark specific energy tending to produce a single or relatively few fragment ions (energyselective dissociation),whereas SID imparts a broad range of internal energies leading to numerous fragment ions. The lack of a large data base on photochemistry/photophysics of large complex ionic systems seriously hampers the development of analytical studies. Photodissociation, due to the more complex instrumental requirements and the neceasi of extracting a small signal from a relatively large backgrounx may never develop to the state of CID; however, for fundamental studies of ionic systems photodissociation may be the method of choice. This is an area that will benefit from extensive study and, due to the limited number of laboratories involved, will require a lengthy period of time to develop.
E. PEPTIDES AND PROTEINS 1. General Considerations. It was clearly recognized in our review 2 years ago (B2) that as a communit we had in hand for the first time the new ionization methdologies and required ion optical systems essential to tackle successfully the classically laborious, demanding, and in many cases inaccessible problems in protein structure determination. However, the fact was that no single laboratory worldwide yet enjoyed the availability of this instrumental arsenal to begin the learning curve necessary for their eventual optimal use in solving such difficult problems. Fortunately each of the ionization methods of importance for pe tide and protein analysis depends upon fundamentally gfferent physicochemical phenomenologies possessing different strengths and weaknesses. Hence, each is uniquely effective for analysis of biologically active preparations of samples with particular properties covering the gamut of disparate physicochemical pro erties, such as extreme hydrophilicity, hydrophobicity, acigty, basicity, or global heterogeneity. A case in point concerns studies of membrane-bound glycoproteins which are resistant to certain types of degradative procedures, and even resistant to immediate success in initial attem ts to use these powerful new ionization methodologies an! instruments. There must be thousands of such structurally complex macromolecules. One such example which has challen ed one of these authors and other mass spectrometrists as we! has been the scrapie prion protein ( E l , E2). There is another compelling reason why all of these methodologies and advanced instrumentation should be made available in a number of centers of existing protein mass spectrometric expertise. This involves the practical but overriding important issues of turnaround time of sample analysis and the sustained use of sufficient instrument time to deal effectively with the numbers of samples which must be run in order to solve difficult biomedical problems. This also involves scheduled, routine access to an instrument to minimize the loss of “irreplaceable (labor intensive preparation)” microsamples. Prevention of loss of precious fractions at the picomole level requires the handling of small volume solutions to minimize time dependent surface absorption and hence, immediate mass spectral analysis. This phenomenon is well-known in protein laboratories (A4,A5, E3-E5). With these notions in mind, an experienced, fully equipped labo474R
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ratory would now be able to make rapid research progress not technically possible a couple of years ago. The level of interest in this methodology is rising rapidly in many laboratories having no previous experience with mass spectrometry (E6), but extensive experience with peptide synthesis ( E n and protein sequence analysis. Therefore, it seems worthwhile to review here where all of these methods stand with res ect to each other in the context of the needs for peptide an! protein identification and structure elucidation. Field desorption (E8) and plasma desorption (E9)methods were the fiit able to measure the molecular weights of polar, chemically and thermally labile substances such as peptides during the 1970s. The fission fragment, event-counting time-of-flight technique (PDMS) soon became dominant, despite its poorer mass measurement accuracy, due to the fact that it was able to analyze higher molecular weight substances readily (up to 20000-30000). The next discovery revealed that olar, labile substances could be sputtered off viscous liquifsolution surfaces (EIO). This was reported in 1980 and rapidl became the most versatile method ca able of covering a simiL mass range. Ita success can be attriguted to the fact that the ion currents produced were very hi h compared with PDMS, and hence spectra could be acquiretf in a few minutes using existing quadrupoles, and single- and double-focusing mass spectrometers ( E l l ) , capable of both unambiguous nominal mass accuracy as well as exact mass accuracy in the case of doublefocusing instruments. LSIMS ion sources could be retrofitted and utilized. A prime characteristic of these methods is their ability to roduce protonated or de rotonated pseudomolecular ion geams with low internal (viironic) energy content-so-called “soft” ionization methods (E10,E12). This fact led to the natural development of tandem arrangements of these ion optical systems (E13, E14) so that vibronic energy enhancement of mass selected molecular species could be carried out one maas at a time with the concomitant recording of so-called collision-induced dissociation (CID) s ectra unique to each mass selected. These tandem metho& have the advantage of being able to tackle the individual com onents of mixtures directly (El@, while at the same time eiminating virtually all of the matrix-associated chemical noise. The liquid matrix s uttering technique was dubbed “FAB” or “liquid SIMS”, &pending upon whether an unfocused neutral xenon atom or a focused cesium ion beam (E16) is primary beam (EIO), used for sputte species from the surface layer of the viscous liquid matrix. %e essential point is that these techniques are dependent upon the surface activity of the sample in the given viscous liquid matrix (E17). Thus components of mixtures are sputtered according to their ability to compete for the surface layer some 50 A thick. Therefore, quantitation of any given com onent requires preparation and use of a stable isotopically &beled analog (E18). In addition, detection of all components in a mixture of peptides in practical terms is not possible because of differences in the hydrophilicity/ hydrophobici ratio am0 st the components of the mixtures. The most hy rophobic 3 1 be sputtered preferentially first in a temporal sense. Hence, if one wishes to find “all” of the components in a protein digest, it is necessary to sort these components as a function of their hydrophilicity/hydrophobicity ratios so that coeluting components have a reasonable chance of being sputtered with similar efficiency. This outcome is readily achieved by reversed-phase HPLC and subsequent introduction of each fraction batchwise into the liquid SIMS ion source of the mass spectrometer. Even this batch method suffers from problems with the most hydro hilic and hydro hobic components. While single amino acds and dipeptid)ea may elute in the void volume and be lost, other early eluting very hydrophilic components may be made sufficiently surface active for sensitive analysis by derivatization with a hydrophobic moiety, such as esterificationwith hexanol (E19). The other extreme representing the most hydrophobic may not elute from reversed-phase HPLC at all, or may not be soluble in a particular liquid matrix and hence, fail. Transmembrane streches of integral membrane proteins may provide such examples (E20). This problem is more readily addressed using other ionization techniques discussed below. There are many similar interface arrangements which permit coupling of liquid chromatogra hic columns to sputtering-type ion sources (so-ca~ed ow FpAB” technique) (1339,
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but in general terms the dynamic range and the detectability of most components is less readily achieved using this on-line approach than b carrying out fraction collecting and batch analysis d a b o v e . In addition, there seems to be a limit of mass size at about 2000 Da for the LC flow probe techni ue, and hydrophilic components such as glycopeptides ten1 to go undetected. Happil , this situation has now changed dramatically with the aivent of electrospray ionization. Electrospray ionization involves the nebulization of a solution of the sample into microdropleta, with their subsequent desolvation into individual charged molecules. These molecules bear either residual protons, permitting detection in sitive ion mode, or have lost carboxyl rotons, for example, detection in the negative ion mode, iepending on the pH of the particular solution being nebulized and the number of basic or acidic residues in the molecule in question (2321).This method is fundamentally different from LSIMS and appears to be relatively independent of the suppression effects observed when different types of components are present in the solution. For example, hemoglobin under certain conditions yields both the a-and &chains independent of each other and the heme moiety as well (E22). However, under conditions where the complex is stable, it is also possible to see ions which are representative of the myoglobin complex (E23). To a first approximation electrospray ionization provides a method for the sampling of solutes which is a reasonably faithful representation of the solution composition, so that detectability seems associated for the most part with the inherent need for com onent solubility and suitable proton affinity. One ma‘or afvantage of electrospray concerns the fact that it is the iirst ionization method in the history of mass spectrometry which is ideally suited to coupling of LC and MS (E24-826). In practical terms, detection of “all” components in a complex mixture is readily carried out by LCeledrospray MS, except for detection of very small hydrophilic components, such as single amino acids or dipeptides which can elute in or near the void volume, and components which are not soluble in the solvent gradient employed. It is now certainly the method of choice for mass mapping of protein digests, since one can easil “see” very large com onents as well, including N- and 0 d e d lycopeptides. d e as noted above, under these elution con&tions very hydrophobic proteins, or h drophobic components of digests, may be lost due to either Lck of solubility or to ag egation or adsorption problems, this is not an inherent rogem with this ionization phenomenon per se. Suitably 801uhzed hydrophobic proteins may be run in their appropriate solvent systems, including integral membrane proteins such as bacteriorhodopsin (E27). The accuracy of mass measurement with electrospray is very good (to 1-2 Da throughout the mass range to 100000 Da) and can be improved considerably by employing image enhancement algorithms (E=) or by use of a double-focusin maa spectrometer capable of accurate mass measurement an higher mass resolution (E29). In addition, using double-focusing instruments the charge state of the species can be determined directly from the isotope peaks. In accomplishing an initial mass surve of a protein or a complex digest mixture of ptides (A13,230),or attempting characterization of hydropfobic systems, high mass proteins (>lOOOOO Da), and proteins with components varying wildly in their physicochemical properties (again the scrapie glycoprotein is a reasonable case in oint vide supra), matrix-assisted laser desorption (maLD) (131)comes into ita own. Very hydrophobic or very large proteins and complex systems may be readily analyzed by matrix-assisted laser desorption if they form a solid solution with the matrix employed. In addition, this method enjoys a major advantage in that it is much more tolerant of the t es of additives, buffers, and salts that are prevalent in biocgmistry labs, which are necesElary to preserve enzyme or other biological activity. One difficulty is SDS, which must be eliminated exce t with 2,5-dihydrobenzoic acid matrix (E32). Matrix-assistedLer desorption is also capable of inherently higher sensitivity than other ionization methods thus far, which makes it ideally suited for analysis of ‘all” components of unseparated mixtures of peptides (A13, E33) or carboh dratea (EM, E35). However the method employing TOF anelyzers suffers from less accurac of mass measurement and inferior mass resolution. As wi& d methodologies, mass measurement ma be optimized by practitioners having a detailed technical undkrstanding of the method with the use
E
d
of ‘proper” [unambiguously characterized standards (E36)] substances for “/time calibration. Mass resolution is the more fundamental problem at the present time, since the technique is susceptible to formation of photoadducts on the higher mass side of the MH+ species, and metastable decay on the lower mass side, not easily dealt with by time-of-flight mass analyzers. Of course discovery of better matrices may alleviate the formation of photoadducts and use of doublefocusing instruments solves the mass accuracy and resolution problems with TOF (A13). Presently with MATD two @pea of instrumental capabilities would be necessary to cover the problems faced in a protein laboratory: the use of energy-correcting reflection systems for measurement of accurate nominal molecular weights at lower masses (as in proteolytic digests), and linear time-offlight for measurement of molecular mass up to the order of 250 OOO Da. Proteins and other macromolecular systems higher than 15000-30 OOO molecular weight have enough internal energy to undergo some unimolecular dissociation during their time-of-flight from leaving the ion source to arriving at the detector, which results in so-called “metastable ion si als”. These signals are not mass resolved, being observe as a continuum from the molecular ion region toward lower mass. Hence, one needs to be concerned about shifts in the centers of gravity effecting determination of molecular mass for two reasons: the formation of photoadducts on the high mass side and the presence of these metastable signals on the low mass side the molecular ions. In summary, each of these distinctly different ionization principles contributes unique aspects to the global detection of the myriad of components present in complex protein systems which, taken in toto, represent an incredibly wide disparity of physicochemical properties. Hence, any lab involved in protein structure/function issues will face analytical problems which M the gamut from “tiny” differences in mass (fl Da) to very large differences or components containing moieties possessing widely differing properties. Taken together this diversity will require access to many of the ionization techniques and the most recent new instrumentation 80 that the unique aspects of these methods/instruments can be called u n as neceasary to assure rapid progress in protein structural g h a t i o n . There is no doubt that use of such mass spectrometric based strategies will result in the reduction of labor costa and the acceleration of discovery of new proteins and characterization of recombinant proteins. This has obvious and compellin benefits for the academic pursuit of fundamental knowleige of the workings of the machinery of cells. From the point of view of rational drug design and issues facing the biotechnology industry, these methods are also indispensible for timely characterization of constructs on the way to recombinant therapeutic agents and to provide quality control for such therapeutics which will satisfy requirements of the U S . Food and Drug Administration. From this discussion it must appear that everythin has been solved, but we must indicate what still needs to be !one. For example, it is still the case that even the best research centers of protein chemistry and mass spectrometry do not have available the minimum suite of these new ionization techni uea and associated instrumentation. ALSO,it is easential that wlen mass spectrometry centers do obtain such equipment, that they take seriously the importance of gaining know-how in matching the sample preparation and handling issues with the mass spectrometric instruments and methods (A5, E3-E5). This is not a trivial point, because one cannot expect the experts in biolo and clinical medicine to become experts in how to success ully assure that their precious picomole of sample is treated wisely in the hands of the mass spectrometrista. For this reason it is incumbent upon the protein mass spectrometry community to get themselves up to speed in how to handle the many ingredients necewary for successful structural analyses of complex proteins. There is just no substitute for extensive experience in this arena, and this is certainly the Achilles heel of the commin ling of this revolutionary mass spectrometric capability and t i e problems of protein research in the life science community. Findy, it is important to purposely set about to design new generations of mass spectrometerswith the highest technically achievable sensitivities, still maintaining the desired other operational characteristics such as sufficient mass resolution and better control over enhancement of vibronic energy
r
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content. As indicated in the Overview, presently there is considerable disparity between the level of sensitivity that can be achieved currently in the best mass s ctrometry labs and that which is achieved routinely in the est biological chemistry labs. The difference is certainly more than 3 orders of magnitude, but can be solved if pursued rigorously by all concerned. 2. Determination of Peptide and Protein Molecular Weight. While it has been inted out repeatedly that mass spectrometry is the m e t h o g f choice for verifying the correctness of structures of synthetic peptides or proteins, it is only recently that the systematic comparative characterization of a 16-mer synthesized in 31 different laboratories has been reported under the auspices of the k i a t i o n of Biomolecular Core Facilities (E7). Both electrospray and plasma desorption mass spedrometric methods were employed as well as capillary electrophoresis in characterizingthese producta. It was noted that a quantitative separation technique coupled with mass spectrometric analysis, such as HPLC and electrospray mass spectrometry, provided the most accurate approach for examining the purity and authenticity of synthetic peptides. It was also noted that while there was generally ood agreement among electrospray mass spectrometry, HPL ,and ca ill zone electrophoresis in the analyses, it appeared that $D% selectively ionized rotected peptides in the presence of unprotected peptides,peadingto an underestimate of the content of the desired product. It was also found that the nature of the modifications observed point to problems which occurred during cleavage, rather than during the actual synthesis. Hence the relatively mild conditions of the Fmoc strategy gave better results, in general. This study also showed that based on the electrospray mass spectrometry, 20% of the samples synthesized in these laboratories did not contain detectable amounts of the intended peptide sequence, despite the fact that these samples all had the correct amino acid compositions and some had full biological activity. The authors conclude that a far greater proportion of the Core Facilities require routine access to either electrospray or other mass spectrometric techniques in order to m i n i possible im act of incorrect and seriously impure synthetic peptides on iological research. This report is obligatory reading. While it would be preferable to anal e synthetic peptide reparations by LC/ES/MS as noted agve, continuous-flow iquid matrix s uttering methods have been commonly used (B37,E37-E397. In addition, both plasma desorption mass spectrometry (E401 and MALD (E34) have been employed, bearing in mind the possible error in mass measurement of approximately 1mass unit with these time-of-flight techniques if they are not properly calibrated. PDMS is clearly less able to provide unambiguous analytical information when dealing with larger intermediates in protein synthesis (E41). Mass spectrometry continues to be the most convenient and effective method for determining the nature of covalent modifications to peptides and proteins. Recent examples include the identification of N-terminal blocking groups including acetyl on x lanase 0342);recombinant tobacco mosaic virus coat protein 6 4 3 ) ;mullet white skeletal muscle myosin alkali light chains (E44); non-mammalian alcohol dehydrogenases (E45);bovine lens aldose reductase (E&); isoforms of cardiac (E47,E48)and nurse shark liver (E49)fatty acid binding proteins; pyroglutamyl of crustacean hyperglycemic hormone (E50);porcine heart aconitase (E51);the heavy chain of both native and recombinant human he atocyte rowth factor (E52);rat seminal vesicle secretion If protein TE53);and NJV-dimethylproline of histone H2B from erythroc es of the marine worm Sipunculus nudas (E54). Exen in-3, a new pancreatic secretagogue isolated from Heloderma horridum venom was found to contain a carboxylterminal amide (E55). Studies of soluble CD4 having reduced binding capacity for the HIV-1 envelo e glycoprotein gp120 proved that asparagine-52 is deami&ted (E56, E57). Mass spectrometry was used to establish the site of signal pe tidase action of a new procholecystokinin-derivedpe tide, 8CK-83, with a sulfated tyrosine and an amidated carioxyl-terminus, suggesting an order of posttranslational modifications that ive rise to the various molecular forms of cholecystokinin (858). From determination of the molecular mass of the lantibiotic Pep5, it is suggested that the maturation of the peptide antibiotic is initiated by selective dehydration of hydroxy amino acids
ge
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478R ANALYTICAL CHEMISTRY, VOL. 04, NO. 12, JUNE 15, 1992
(E59). Mass spectrometric investigation of bovine proopiomelanocortin revealed that the heterogeneity was due to both artial sulfation of tyrosine-28, and partial N-terminal pyroghmation (ESO). Measurement of the molecular weight of the subunit of NADH:ubiquinone oxidoreductase from bovine heart mitochondria by electrospray ionization established the resence of covalently attached pantetheine-4'phosphate b 6 1 ) . The successive addition of glutamyl units on the y-carboxyl group of glutamate-445 of a-tubulin was established by mass spectrometric analysis (E62, E63). S(1,2-Dicarboxyethyl)cysteinewas found as position 77 in a mutated human lysozyme expressed in yeast (E64). Upon mutation of Cys-77 to Ala in this system, two proteins with different specific activities were secreted by yeast. Mass spectrometry was used to establish that one of the mutants was covalently modified by glutathione at Cys-95 (E65). Mass spectrometry has been used in several ways to study protein prenylation. Initial degradation of S-prenylcysteine derivatives was carried out with h e y nickel with sub uent identification of the hydrocarbon fraction by G C / 8 , including the farnesylation of the y-subunit of transducin (EM), the ESMS characterization of intact farnesylation of peptides of transducin y-subunit (E67),prenylation of cytosolically and proteins in HeLa synthesized proteins of CHO cells (EM), cells (E69). Earlier, tandem mass spectrometry was used successfully in the characterization of S-farnesylated peptide isolated from the a-factor of Saccharomyces cereuisiae required for sexual conjugation (E70). Further work on characterization of the S-prenylation of cysteine peptides has been reported (E71). This work reemphasizes the advanta es of dealing directly with a covalently modified peptide by uid SIMS and preferably MS/MS, rather than resorting to %eg radation, reduction, and isolation of the mixture of hydrocarbons for GC/MS characterization. Studies of a variety of proteins have revealed palmito lation of isolated cysteines, as well as the modification of ac$acent cysteines flanked b proline residues near the N-termini of surfactant associatdproteins. Myelin proteolipid protein was while Band 3 shown to be palmitoylated at Cys-108 (E72), rotein in human erythrocyte membrane was modified on ys-69 (E73). Pulmonary surfactant proteins isolated from porcine (E74),bovine (E75),canine (E76, E77), and human (E75) sources have been shown to be modified by two palmitoylyl groups on adjacent cysteine residues near the Nterminus of the protein flanked by proline residues. Using a combination of chemical and proteolytic digests and mass spectrometry to analyze the covalently modified peptide, it was established that His-93 was modified by the CClz moiety derived from xenobiotic bromotrichloroethane of myoglobin during metabolism of the xenobiotic (E78). Studies of crustacean hypoglycemic hormones have been extended to characterization of neuro ptides from the lobster Homarus americanus (E79, E80). Eructure elucidation of a new 5 kDa peptide from neurohaemal lobes of the corpora cardiaca of Locusta migratoria turned out to be rich in alanine residues (E81). Evidence has been obtained for a relationshi between bombesin and gastrin-releasing peptide ( E 8 8 Studies of the degradation producta of biol ' d y active a-bag cell peptide suggests the resence o leucine aminopeptidase-like activity in A p g s i a hemolymph (E83). Confirmation of the sequence of pituatary human galanin was carried out by PDMS measurement of its molecular weight (E84). Characterization of posttranslational processing of canine intestinal cholecystokinin-58 revealed the lack of its carboxy-terminal nonapeptide (E85). Variant antithrombin Rouen-IV with reduced heparin affinit was shown to have cysteine substituted for Arg-24 (E86). qhree forms of insulin-like growth factor 11have been isolated from human Cohn paste IV of human plasma (E87) and insulin-like growth factors I and I1 from porcine plasma (E88). Electrospray continues to be a very important method for verifying molecular weight and assessing purity of recombinant proteins, including interleukin 2 and interferon y (E89). Alternatively, liquid SIMS and PDMS have been employed with human interferon a-2b (E90), interleukin 18 (E91,E92), and human interleukin 6 (E93). PDMS was used for the analysis of recombinant human parathyroid hormone (E94). A variety of C-terminally truncated derivatives of recombinant hirudin (E95)have been characterized by liquid SIMS,as well as a desulphatohirudin (E96).Baculovirus recombinant
6
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MASS SPECTROMETRY
pre rocecropin A was shown to be correctly processed, incluing removal of si nal eptide and pro-part, with biologically active and ami ate cecropin A exported into the hemolymph 0397). Highly cationic polypeptides called bactenecins isolated from large ranules of bovine neutro hils which exert otent antimicrotial activit in vitro have een characterhexby PDMS (E%). Cuttlefd sperm protamines have been shown to be among the most basic proteins ever characterized, with an arginine content of 77% (E99). Characterization of thioredoxin from green al a, Chlamydomonas reinhardtii was characterized by P MS (E100). PDMS was also employed in the characterization of a major protein component in the light harvesting complex of the green photosynthetic bacterium Chlorobium limicola f. thiosulfatophilum (E101).The characterization of phototoxic lipodepsipe tides from Pseudomonas syringae pv. syringae have been ctaracterized by NMR and liquid SIMS (E102). A variety of cyclic hepatotoxins from cyanobacterium Nostoc (E103) and Mycroc stis aeruginosa, Microcystis uiridis, and Microc stis Luesenler i t (E104) have been characterized by liquid &MS and NMgiE103, E104). Some of these toxins are inhibitors of protein phosphatases, making them important biological tools. Elucidation of the structure of a specific inhibitor of bacterial protein biosynthesis acti on elongation factor Tu was shown to belong to the thiazoly pe tide group of antibiotics (El&). A potent glycoprotein b - d a a n y and platelet gregation inhibitor, decorsin, has been c aracterized from 8 e leech Macrobdella decora (E106). Two C-terminally truncated forms of aprotinin have been charaterized from a reparation of bovine aprotinin by PDMS (E107).PDMS has een used to confirm that bovine pancreatic thread protein contains two disulfide-bonded olypeptides, and an A chain of 101 and a B chain of 35 resizues (EIOB).It is noted that the rotein is present in ancreatic secretion, brain lesions m Aeheimer’s disease an?I Down syndrome, and regenerating rat pancreatic islets. The primary structures of metallothioneins have been reported for the oyster Crassostrea.uirginica (E109), and the domesticated duck (EIIO).Liquid SIMS and PDMS were em loyed to characterize isoz mes of dihydrodiol dehygogenase (E111).Liquid S I h S has been employed to establish that the active site of 1-aminocyclo ropane-lcarboxylate synthase is inactivated by its su strate, Sadenosylmethionine (E112).It was shown that an active site lysine residue was alkylated by the 2-aminobutyrate portion of the substrate. Liquid SIMS was em loyed to confirm over 90% of the sequence of a-fetoprotem &113). Elucidation of the chemistry of 4-aminobutyrate aminotransferase by the antiepileptic drug, vigabtrin, has been re orted (E114). Identification of a glycosyl group covalently inked to Asp-6 in an a-glucosyltransferase from Streptococcus sobrinus implicata the aspartic acid as the residue involved in stabilizing an oxocarbonium ion transition state (E115).Structural differences between the A and B chains in the I11 CS region of human plasma fibronectin have been established by liquid SIMS (E116).Limited proteolysis and mass ma pin of an immobilized antigen-antibody complex was usezto ifentify molecular epitopes (E117,E118). The results for both human and guinea pig anaphylatoxin C3a complement have been reported (E119). Documentation of the cleavage site of the amyloid precursor protein in Chinese hamster ov cells leading to secretion was carried out by PDMS (E12O).%udies of herpesvirus maturational proteinase, assemblin, established the assembly protein precursor cleavage site (E121). The identification of neutrophile elastase homologues with strong monocyte and fibroblast chemotactic activities have been characterized in porcine and human sources by PDMS (E122). The com lete amino acid q u e n c e of the anti-a(2-8)- lysialic acid anti!od mAb735 light chain has been repo+$E123). Li uid SIMJhas been employed for mappmg stram variation an1 posttranslational modifcations of Newcastle disease virus nucleocapsid proteins (E124, E125). 3. Disulfide Linkages. A variety of mass spectrometric-based strategies have been applied to the problems of assignment of disulfide-linked proteins, including digestion with proteases of differin cleavage specificities, chemical cleavages and artial acidf hydrolysis, and both low- and high-energy co&ion-induced dissociation of the cross-linked dipeptides. These are illustrated using plasma desorption
i f
1
b
1
i
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mass spectrometry (E126), li uid SIMS (E127), electrospray (El%),and collision-induce1 dissociation (E129). Proteins which have been studied include a small protein from barley, a hi hly basic glycoprotein from porcine platelets, human insufin (E126), egg white lysozyme (E127), rat C-reactive protein (El%),recombinant human growth hormone rece tor (E130), recombinant human relaxin (E131),natural antrecombinant insect defensin A (E132), AQN-3, a carbohydrate binding protein from boar sperm (E133), a-amylase inhibitor (E134), human prostatic acid phosphatase (E135), albolabrin, an RGD-containing peptide from the venom of Trimeresurus alborlabris (E136). Preliminary results have been presented on the identification of thiols and disulfides in peptides and proteins usin HPLC with a modified mercury Hg/Au electrochemical tetector and liquid SIMS (E137). In addition, using reduction and alkylation plus electrospray mass spectiometry it has been shown that accurate counting of cysteines, disulfide bridges, and free sulfidal groups may be mapped in proteins (E138). 4. Electrospray. Electrospray ionization has clearly emerged as the method of choice for the accurate measurement of the molecular weights of proteins, and the discovery of the nature and extent of the covalent modification by posttranslational processes, xenobiotic agenta, and recombinant expression systems. In addition, it is certainly a major tool in quality control in the biotechnology industry, taken together with its LC/electrospray on-line analytical capability. It is also the method of choice for the detailed characterization of very complex mixtures of protein digests. Studies of intact proteins and glycoproteins and their recombinant analogs have been discussed (E139, E140). The advantages of such techniques for very basic proteins containing more than 50% arginine have been well illustrated (E141). Electrospray has also quickly become a routine clinical tool for the diagnosis of abnormal or variant hemoglobins (E142-El45). It has been also used for rapid analysis of the proteolytic products of a model antigen, Staphylococcus aureus nuclease, treated with an acidic endoprotease,cathepsin D, and asaessed for antigenicity using a T cell recognition assay (E146).More recently, electrospray has been used to identify peptide antigens resented by class 1and 2 major histocompatibility molecJes from cell culture (A7,AB). Adducts of the electrophilic metabolites of acetaminophen and benzene with human hemoglobin have been detected by electrospray (E147). Neuro anin, a brain-specific in vitro substrate for protein kinase fshows the immediate power of the method in revealing two components in a preparation purified to homogeneity from bovine forebrain (E148).The nature of the heterogeneity has yet to be elucidated, although perhaps represents an N-terminal block and a modified lysine residue. Another example concerns the identification of the C-terminal residue of a 69 and a 67 kDa form of an outer membrane protein of Bordetella. pertussis (E149). Electrospray was used to measure the molecular weight of the overproduced C-terminal domain of 6-deoxyerythronolide B synthase of Saccharopolyspora erythraea and showed that the urified protein had not been posttranslationally modifie by attachment of a 4’-phosphopantetheine group (E150).Electrospray measurements were also indispensable in monitoring the chemical modification of the protein active site by the serine proteinase inhibitor phenylmethylsulfonyl fluoride (E151).Electrospray measurement of the molecular weight of FK506 and rapamycin binding protein, FKBP, isolated from calf thymus, was in close agreement with the calculated m898 for the proposed primary structure, confirming the linear sequence, C-terminus, and eliminating the possibility of glycosylation or other modifications (E151). It should be noted that a recent review of electros ray ionization contains a table of peptides and proteins which L v e been analyzed by the technique, to ether with notes and references (see Table IV, B53, E151b7. One of the more interesting reports follows the observation of the intact myoglobin complex noted above ( E D )for the study of noncovalent enzyme-substrate and enzyme-product complexes using hen egg white lysozyme and ita substrate hexasaccharide of Nacetylglucosamine (E152). This initial report suggests many interesting opportunities for probing noncovalent binding interactions involved in molecular recognition. In this study the relative ion intensities of the substrate-enzyme complexes qualitatively parallel the known association constants in so-
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ANALYTICAL CHEMISTRY, VOL. 64, NO. 12, JUNE 15, 1992
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lution and suggest that association constants in the order of lo5 per mole are necessary. This raises continuing concerns over the existence and severity of suppression effects in mixtures with these soft ionization methods, as pointed out above. Somewhat less of a surprise, but nevertheless interesting is the observation of an imine intermediate on dehydroquinase by electrospra mass spectrometry (E153). Using a variety of chemical mienzymatic d adations, MALD w~ employed to ma recombinant i n t e r l e g - 2 (E154). Using ribonuclease A a n i B as model rotein and glycoprotein, the protease clostripain was usec r to study the time course of digestion. Matrix-assistedlaser desorption was used to detect the nature of the proteolytic fragments which a peared as a function of time. From this data, sequence-ortfered digest maps were obtained with inherent sequence overlap information (E155). Taking advantage of the very wide mass ranges available with both electrospray and MALD techniques, use of cleavage techniques for low abundance amino acids and proteins, such as Met, Trp, Cys, and His, has recently been reported (E156). Horse heart myo lobin and plasminogen activator inhibitor were used as motels in these studies. The types of artifacts produced can be a serious problem, even with the simplest of these methods, such as cyanogen bromide cleavage. This is only the beginning of attempta to understand such chemical digests of lar e proteins, particularly when these proteins contain disulide linkages, as noted in this paper. Clearly, separations and subdigestions followed by collision-induced dissociation analysis would be very valuable in determining the nature of the chemical reactions and the types of products formed. Studies by MALD of histidine tRNA synthetase expressed in E. coli have shown part of the C-terminus to be missing (E157). In addition, MALD readily showed that the anticoagulant peptide was phosphorylated and that the aspartate receptor expressed in E. coli contained four minor com onents and a missing N-terminal amino acid (E157). StuPes of intact monoclonal antibodies and their analogs con'ugated with chelators and anti-cancer drugs have been andyzed by MALD (E158). In addition, estimates of the mass of carbohydrate present in the monoclonal is judged by the mass difference between the MALD data and the calculated molecular weight of the rotein from cDNA sequence. Further measurements after redktion of disulfide linkages ave mass values for the light and heavy chains. Recently, d A L D has also been used to assess the multimeric states of proteins by using glutaraldehyde as a crosslinking agent to preserve protein subunit association complexes present in solution ( E159). 5. Phosphorylation. In many cases measurement of the molecular weight of a peptide or protein, or measurement of the molecular wei ht of a peptide in conjunction with Edman sequence data w!i establish the site of phosphorylation of a protein (E160). Phosphorylation of tyrosine 67 in bovine brain myelin basic protein was established using the protein tyrosine kinase p561ck(E161). Serine 472 was identified as the site of phosphorylation in pp64 from human cyclomegalovirus (Towne) (E162). A combination of liquid SIMS and Edman degradation localized phosphate moieties to Ser-3, Ser-99, Ser-112,and Ser-116 in salivary cystatin SA-I11(El63). Use of electrospray to measure the molecular weight of sea urchin sperm-specifichistone H1 showed the presence of seven, eight, and nine phosphate groups (El&). An endoproteinase Lys-C digest and liquid chromatogra hic electrospray mass spectrometry experiment on the a-suiunit of nicotinic acetylcholine receptor contained fractions showing three phosphorylated sequences (E165). Studies of differences in peptidoglycan composition ap arent between parents and amp operon mutants containeamonomeric and dimeric pentapeptides, respectively (E166),which allowed the identification of the f i t genes which control peptidoglycan composition in response to the environment, and the coordinate regulation of 8-lactamase induction in peptidoglycan composition by the amp D operon. The structures of pyoverdins Pt, siderophores of Pseudomonas tolaasii NCPPB 2192 and pyoverdins Pf of Pseudomonas fluorescens CCM 2798 possess, at the N-terminal end of their peptide chain, a chromophore derived from 2,3-diamino-6,7-dihydroxyquinoline(E167). Location of modified tyrosine in chitinase inactivated by l-ethyl-3-(3(dimethy1amino)propyl)carbodiimide has been reported (E168). Studies of the number of functional lipolyl groups 478R
ANALYTICAL CHEMISTRY, VOL. 64, NO. 12, JUNE 15, 1992
in dihydrolipolyl acetyl transferase of the pyruvate dehydrogenase multienzyme complex from E. coli has been carried out by a combination of covalent modification with N-ethyl[2,3-l4C]maleimide by electrospray mass spectrometry (E169). 5. Sequence Determination by Tandem Mass Spectrometry. Most of the work during this period was continued using one of two distinctly different strategies, sometimes referred to as low-energy and high-energy collision-induced dissociation. This distinction is based on the kinetic energies of the selected beam in MSI, being either in the order of 50 V for quadrupole instruments, or 8-10 kV in four-sector double-focusing instruments. These different experiments deposit substantially different quantities of internal energy into the selected beam, thereby, in the low energy case, resulting in the induction of fragmentation in the peptide backbone, and in the high-energy experiment producing these ions and additional fragmentation in amino acid side chains and other moieties, and an array of immonium ions of either single or covalently modified amino acids. The internal energy available in the high-energy experiment has been estimated to be in the order of 12-15 eV, whereas in the low-energy experiment the energy is in the order of 2-3 eV corresponding to 23 kcal/(mol eV). Hence, there are some fundamental differences, in t at in the low energy case the isobaric pair leucine/isoleucine are indistinguishable, and in many cases there is incomplete information regarding sequence from a single experiment. This situation requires the preparation of two or three chemical derivatives, such as the acetate or the methyl esters and the recording of low-energy CID spectra on those derivatives as well. In the high-energy case, usually the information necessary to determine the sequence is present inherently in the high-energy CID spectrum. There are other practical differences in the use of these two methodologies. In the low-energy case, high sensitivity may only be obtained by allowing molecular species with several mass unit-wide window into MSI. The high-energy case can employ multichannel array detection systems and is routinely capable of selecting only the C12 molecular ion isobar, thus providing a monoisotopic CID spectrum with unambiguous determination of nominal mass. This information in the low-energy case is obtained by mass shifts from the multiple derivatives which must be prepared for recording CID spectra independently. These points are based on qualitative comparisons of spectra of the same peptides and oligosaccharides obtained under low-energy and high-energy CID conditions (El 70, El 71). It should be noted that work has continued on derivatization of peptides aimed at placing a permanent charge on either the N- or C-terminus to enhance remote site fragmentation processes. These include studies of the ethyltriphenylphosphonium moiety (E124,E173). Also, the additional hydrophobicity is probably responsible for the increase in sputtering efficiency observed. Initial work on studying the fragmentation processes of the triphenylphosphonium derivative has been presented (El74). Using stable isotope labeling, collision-induced dissociation processes of protonated and metal-cationized peptide8 have revealed interesting intramolecular rearrangements in the gas phase (El 75-El 77). A comparison of mono- and diprotonated species of peptides containing modified amino acids, particularly those that involve arginine or other highly basic sites, and subjected to low-energy CID were found to produce extensive side-chain cleavages superimposed on sequence ions in the monoprotonated case. These results are difficult to interpret with a relatively low degree of coverage of the complete peptide sequence, in contrast to the CID of the doubly-protonated chain contained in a complete characterization of the overall sequence of the peptide ( E l 78). In the presence of lysine-specific fragments in liquid SIMS spectra found 16 Da below common acylium, a B-type ion has been reported (El 79). The conversion of asparagine-199 into isoaspartate in bovine and porcine somatotropins by incubations in neutral or alkaline DH was established bv low-energy CID analysis (E180). Asimilar deamidation 'In tumor necrosis factor was solved using high-energy CID analysis as well (B55).
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A variety of peptide and protein sequencing problems have been reported usmg low-energy CID methodology, including a new pe tide in the FMRF amide famil isolated from the CNS of tie hawkmoth, Manduca sexta (&82); identification of proctolin in the CNS of the horseshoe crab, Limulus polyphemus (E182); and a diuretic peptide from the tobacco homworm, Manduca sexta (E183).In addition, the complete sequence of the acidic subunit from Mojave toxin (E184), muscle parvalbumin (E185),hosphorylation site in neurons ecific class I11 @-tubulin&186) and polyglutamation of &u-438 in ita C-terminal uence (E186)have been reported. Thii method was used toyentify threonine-97 as the site of specific phosphorylation in bovine myelin basic protein b mito en activated protein kinase (E187).Studies of s inaci ljht%a&ating chlorophyll protein 11established the icfentity o three different N-terminally acetylated phosphopeptides (E188). Us' electrospray and collision-induced dissociation of the (M +%)3+ ion of a peptide in F-actin cross-linked by N(4-azidobenzoy1)putrescineestablished that lutamine-41 is intermolecularly cross-linked to Lys-113 (i189). Further studies using the tri-protonated species identified the phoshorylation sites in pp42/mitogen-activated protein kinase PE190). Several laboratories have discussed collision-induced dissociation spectra of multiply-char ed peptide ions produced by electmapray ionization using bo& singlequadrupole (El91) uadrupole (E192, E193)instruments. Less freand quently, C studies have been carried out on hybrid tandem instaumenta (E194).Identification of a pyroglutamyl peptide derived from chromogranin B has been reported (E195).The sequence of adi kinetic hormone which controls lipid metabolism in a d g and carbohydrate metabolism in larvae of Manduca sextu was determined b a triple-sector instrument (E196). A charged adipokinetic-iormone-familypeptide in the fruit fly Drosophda melanogaster was sequenced by tandem hybrid CID analysis (El97). Capillary HPLC/FAB mass spectrometry was used to confirm the rimary sequence of a-crystallines from bovine lenses, and &D analysis established the site of serine phoshorylation (E198).CID analysis was important in estabishing the site of covalent binding of the lipase inhibitor tetrahydrolipstatin to the putative active site serine of pancreatic lipase (E199).Low-energy CID analysis of a nonapeptide of apoB-100 showed the oxidation of cysteine-4190 and methiome-4192. It is presently not known whether there is contribution from artifactual oxidation from these experiments (E2WE202).The reactivity toward peptides and proteins of S-(N-methylcarbamoyl)glutathioneto a glutathione con'ugate of methylisocyanate and its correspondent cysteine adduct was characterized by FAB and collision-induced dissociation analysis. These studies establish that free sulfhydro on peptides a ar to be prime targets of covalent modification as the N-methylcarbamoyl)functionality at internal cysteine residues (E203). 6. High-Energy Collision-Induced Dissociation. High-energy collision-induced dissociation spectra contain immonium ions as well as acyl and immonium dipeptide ions which aid in the establishment of amino acid composition in the interpretation of this type of spectra. Recently a tabulation has been made of immonium ions and the modified residua which they compond to, as well as related ions which are usuall observed in these spectra (E204).These highenergy CI spectra contain large numbers of monoisotopic ions occurring at discrete, unambiguous nominal masses. Extracting all of the structurally related information from such spectra can be, in many cases, very time consuming. The MI" group (E205)has developed computer-based a$orithms that are designed to correlate observed fragments wlth those redicted from postulated sequence, taking into consideration Enown empirically-derived fragmentation processes. More recently, the UCSF group (E206)has adopted the alternative strategy of searchin for patterns due to ion families based on known empirical gagmentation processes. For each family detected, a so-called "y-center", a subsequent search is conducted for connectivit among * centers" to establish sequence possibilities. Jhese are tren scored by a variety of criteria. The final selection of a "correct* sequence interpretation is left to the researcher. This strate lends itself to the ready detection of moieties not containeTin the com-
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mon amino acid mass residue tables and is particularly useful in interactive spectral interpretation. The al orithm is commercially available to run on a Mac 3-basef Sparc Station (E207). Work continues aimed at understanding the structural and kinetic basis of fragmentation under high-energy collision conditions including the effect of proline and proline-conthe differentiation of hydroxyproline taining peptides (E208); and rocesses uni ue to N-terisomers and isobars (E209); minal glutamine (E210).Severaflaboratories Rave utilized the proteolytic digestion of proteins in '*O-containing water for the purposes of location of the carboxyl terminus of the E212)or assignment of C-terminal sequence protein (E211, E214). Further work on the formation of y-type ions (E213, ions has been presented (E215).There is also considerable interest in the fr mentation processes of sodiated peptide species and pepti e amides (E216)as well as fragmentation of backbonemodified ptides (E217) where the normal amide linkages are replaced groups such as thiomethylene ether, thioamide, methyleneamine, and thiomethylene sulfoxide moieties. As discussed in earlier sections relating to low-energy CID studies involving the ethyltriphenylphosphonium derivatives, laboratories interested in high-energy processes studied the trimethylammonium acetyl moiety attached to the N-terminal E219). The CID spectra of amino group of a peptide (E218, these derivatives are simple, exhibiting the same characteristics as peptides bearing stron ly basic arginine at the Nterminus. As has been shown mfier, high-energy CID spectra are virtually ideal for characterizing unexpected reaction products which form during the synthesis and particularly deprotection of synthetic peptides (E220).High-energy CID analysis was also employed to explore the utility of cysteine-specific cleavage reactions together with laser desorption methods (E221). Three sea anemone neuropeptides have been isolated by radioimmunoassa for a carboxyl-terminal sequence and characterized by CK> analyeis and roton NMR. They contain the L-3- henyllactic N-terminal [locking group thought to render t\ese neuro eptides resistant to nonspecific aminopeptidases (E222,5223). A series of hypertrehalosaemic peptides have been isolated from the corpora cardiaca of the cockroaches Leucophaea maderae, Gromphadorhina ortentosa, Bluttellu gerrnunica, and Blatta orientalis and ofthe stick insect Extatosome tiaratum. Their primary sequences were deduced from collision-induced dissociation spectra (E224). Further studies employed CID analysis for the characterization of four allahtatic peptides isolated from the brains of the virgin female cockroach Diploptera punctata. Extraction of over loo0 brains yielded 80-100 pmol of each of these four peptides (E225). The structure of three bombinin-like peptides isolated from the skin of Bombina orientalis were established by tandem mass spectrometry. These pe tides were found to possess potent antibacterial activities, gut no appreciable hemolytic activity (E226).Tandem mass spectrometry has been employed in resolving the differences in the roposed structures for Staphylococcus aureus V8 protease &227). Neither the gene sequence nor the protein sequence of the first 215 amino acid residues were found to be entirely correct. These reaulta agreed with the protein sequence in only four places and with the gene sequence in eight places (E227). The sequence of the N-terminally blocked peptide from cadmium-induced metallothionein in a mollusc, the oyster Crassostrea virginica, is identified as an acetyl group by CID analysis (E228).The amino acid se uence of a protease inhibitor isolated from Sarcophaga bulata was determined by CID analysis. The molecular wei ht of the intact material was measured by both MALD an%liquid SIMS. From the established primary sequence it was clear that the distribution of cysteines was entirely independent of cysteine distribution in other proteases (E229). The se uence of another serine protease inhibitor, ecotin, found in %e periplasm of E. coli, was established by Edman degradation and tandem mass spectrometry. The molecular weight of the protease was determined by both MALD and electrospray mass spectrometry (E230).Using a combination of Edman and tandem ma^ spectrometricmethods, the amino acid sequence of a protein inhibitor of rotein kinase C has been shown to be N-terminally blocket! by an acetyl group
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(E231). This inhibitor does not show significant homology with any other known protein but ap to be a zinc-bmding protein, although the sequence di$" not reveal a zinc-finger structure (E232). Further combined use of Edman degradation and CID analysis has established the complete amino acid sequence of bovine lia maturation factor @ and it also is an N-terminally acetyfated protein (E233). In an interesting study aimed at map ing rabbit lung flavin-containing monooxygenase and the kterminal blocking acetyl group, more than 50 ptides whose molecular weights did not correspond to the MO sequence were observed in reversed-phase HPLC fractions. Approximately 70% of these were sequenced by CID analysis from the mixtures, leading to the discovery that these matched completely a calciumbinding protein, calreticulin. It is pro osed that. calreticulin complexes and stabilizes FMO, althoug! the physiologd role of this complex is not presently known (E234). Us' proteolytic digest mapp' and CID analysis,the core a m y l 3 protein and cerebrovasc ar amyloid protein isolated from the cerebral cortex at autopsy of an Alzheimer's disease patient revealed that, in the case of CVAP, the C- and Ntermini were frayed, A more complex pattern of fraying was observed using MALD on a minimally purified CAP sample from a different patient, leading to the suggestion that the amyloid precursor is subject to abnormal proceasing in persons suffering from Alzheimer's disease (E235, E236). Using both protonated and depTotonated enerfy ?ID analysis, it has been possible to identlfy the posttrans ational modification at two sites in rabbit elongation factor Ia to be glycerylphospho lethanolamine amidation of glutamic acid side chams (E238. CID analysis established the N-terminal acetylated blocking oup of the large subunit of ribulose1,5-bisphosphate carroxylase/oxygenaee from several plant species. It was also im rtant in sequency a trimethyllysine posttranslational mo&cation on one o the penultimate N-terminal tryptic fr menta (E238). Accurate mass measurement usin liquid%MS, N15 derivatization, high-energy CID analysis, &V-visible spectroscopy, and NMR revealed that the active site of bovine serum amine oxidase contains a new redox cofactor, 6-hydroxydopa (E239). The first detailed report of the use of high-energy CID analysis for sequencin throu h cystine bridges of intermolecular disulfide-bond% peptiies has been presented (E240). Fragments due to peptide chain cleavage are observed at lower abundance in the product ion spectra and can be sufficient to sequence both of the disulfide-linked peptides without any prior knowledge of the peptide or protein sequence. In cases where the peptide sequence-related roduct ion yields are poor, the intensities for the disulfide cEav e ions are usually sufficient to determine the molecular weiY lts of the component cystine-bridged peptides. These teckiques were illustrated for synthetic disulfide-linkedpeptides and then applied to human transforming growth factor a, a compact rotein containing 48 residues and three disulfide linkages pE240). The use of li uid SIMS and CID analysis for the. investiation of phosplor ylation sites has been described in detail TE241, E242). Using a strategy of stabilizin transjently phosphor lated aspartate residues on CheY (#243), similar studies oTphos horylated NtrC, a protein phosphatase covalent intermegate that activates transcription, have been re orted (E244). Location of the homoserine product of borotydride reduction of phosphoaspartate is readily achieved from the CID spectrum. It should be emphasized once again that high-energy CID anal sis and tandem mass spectrometry are ideal strategies for ietermining chemically modified residues in proteins. Several different types of examples of this follow. Using acrylodan, which normally modified cysteine residues, derivatization of recombinant interleukin-l@was carried out under native conditions. Two major covalent protein acrylodan adducts were enerated and identified by high-energy CID analysis (E2457. It was found that one adduct was on cysteine-8 and the other on an unusually reactive lysine-103. Neither covalent product altered si ificantly the biological activity, leading to possible use o these modified protein anal0 s as robes of receptor bindin ( E M ) . E oxycreatine, an aftnitygbel of creatine kinase d a t irreversiby and completely inactivates the enzyme, was shown to bind to cysteine-282 in the active site (E246). In studies of biotinylation of gonadotropin-releasing hormone and analogs, it was ob-
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served that substantial biotinylation of residue serine-4, and to a lesser extent tyrosine-5 occurred. This was abolished durin biotinylation under denaturing conditions, indicating that %e 0-biotinylation was dependent on peptide conformation. These results have led to the suggestion that anhydroxysuccinimidebiotin esters are capable of detecting highly reactive hydroxyamino residues (E247).In further work, the use of hemoglobin as a surrogate nucleophile for reactive xenobiotics has been studied in the in vitro reaction of 1,4benzoquinone with erythrocytes (E248, E249). Covalent attachment of polyeth lene glycol to enzymes has been shown to prolong plasma d - l i v e a and improve therapeutic potential of such enzymes. High-energy CID analysis has been employed to locate the sites of covalent modification in superoxide dismutase (E250, E251). In the biotechnology industry, there are many examples of the use of these methods for the detailed comparison of native and recombinant versions of potential therapeutic proteins. One interesting case has centered around characterization of native and recombinant relaxin, including the location of three disulfide bonds (E252, E253). CID analysis was important in establishing the nature of the cyclic imide-containing ptide in a succinimidevariant of a methionyl human growth Krmone occurring at asparagine-130 (E254).
F. OLIGOSACCHARIDES AND GLYCOCONJUGATES
As noted in our previous review (B2),those Components of living systems comprised of carbohydrates as the dominant structural component or even as a single monosaccharide moiety, occur within several major structural motifs in bioseas very different roperties and polymers. As such they presently require a n a F c a l strate ies taigred to each structural motif. In ad ition, since %e biosynthesis of oligosaccharides requires sequential action by glycosidases or glycosyltransferases with different structural specificities expressed in a cell type-dependent manner (as far as we presently know), the occurrence of families of similar oligosaccharide structures is the rule rather than the exception. This situation gives rise to so-called microheterogeneity and compounds the challenging problems of separation of individual structurally homogeneous components. In addition, since little is understood about the presence of a variety of glycosidases in particular cell types, it is often difficult to evaluate whether some of the microheterogeneity might be due to catabolism, rather than incomplete biosynthesis. Because high-quality tools with degradative specificity such as proteol ic enzymes (E5) have not been available, more emphasis as been placed on chemical degradative methods to obtain components for structural characterization (PI). Many of these methods are chemically quite harsh, leading to the generation of multiple components from si le starting materials, thereby compounding the analytic3 problem. While the common monosaccharides involved in possible structural variations of oligosaccharidesnumber far fewer than the 20 amino acids characteristic of peptides, the anomerity and linkage positions 1,2-, 1,3-, 1,4-, and 1,6-, as well as branching configurations, vastly multiply the numbers of possible structures for a given molecular size. The structural goal is to define these parameters unambiguously. For all of these reasons, the microchemical isolation, separation, structural characterization strategies, and arsenal of tools are less well advanced (F1)than in the protein and nucleic acid fields. However, recently it has been suggested that certain substructures, some of them known antigenic determinants for example, may be involved in molecular recognition leading to viral infection and bacterial colonization, and poesibly attracting natural immune cells to cells ex reasing articular surface carboh drate structures. These Endings gave stimulated considerabe interest and excitement (F2) in the field of glycobiology. Examples of such work abound, such as determination of the X-ray structure of hemagglutinin with sialic acid (F3), as well as those using methods of mass spectrometry, such as studies of the binding of the riodontal pathogen Fusobacterium nucleatum to a parotiEaliva gl coprotein (F4). Recent1 established carbohydrate biotecgnology efforts are focusdon the development of insights into carbohydrate biology and potential therapeutic agents, more complex than the well-known model of heparin. One example is the cell adhesion molecules such as the selectin receptor,
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which recognizes the sialyl Lewis X antigen (F2).The devel0 ment of pharmaceuticals based on fundamental structur8knowledge of antigens and receptors leads quickly into the poseibilities of blocking the immune system’s lymphocytes in processes of inflammation, with ramifications for autoimmune diseases such as rheumatoid arthritis, type 1diabetes, etc. While the strategies and tools required for dealing with the man issues in glycobiolo have a long way to go, ma’or c c u r r g In cases where oligosaccharides breahuoughs have o can be obtained by enzymic or chemical release from their lipids or proteins, separation based on their oxyanions on micropellicular quaternary ammonium resins has been a major innovation over the last 3-4 years (F5).This high pH polyanionic chromatogra hy with pulsed amperometric detection has the capacit for cpeanly se arating many of the neutral and anionic, inching sialylatetfphos horylated, and sulfated isomeric forms of oli osaccharides isobkd from lycoproteins or glycolipids. In afdition, on-line membrane iesalting has been shown to be sufficiently effective that reductive amination can be erformed for structural studies by negative ion liquid SIMSand hi h-energy CID analysis (F6,F7). This oxyanion chromatograp% may easily determine, for example, the relative amounts of d e Man, high mannose isomers from a variet of yeast sources (F6-F8), compared with bovine sources &9),includin the various sible branched isomers of the east itaelf (F8f. These h a z e v i o u s l y been chromatograptically unresolved and required analysis of the mass spectral fra entation pattern to detect and establish a revised yeast ano structure (FlO). Having in hand this new oxyanion chromatographic method for the effective resolution and separation of even isomeric isobars, there are presently several strategies for the use of maea spectrometric methods for the structural characterization of putative isomerically pure oligosaccharide isomers. Since oligosaccharides are extremely hydrophilic and do not contain a sensitive chromophore, inclusion of a derivatization step is advantageous in increasing the hydrophobicity necessary to provide the best mass s ectral sensitivity and introducin a suitable chromophore 6 7 ,F11,F12). This ste allows for either extraction from biological media or was in on reverse-phase to eliminate salts and buffers and maten& which tend to degrade the quality of mass spectra (Fll),or even preclude the ossibility of obtaining the spectra, in the case of liquid SIMg. These derivatization strategies are reductive amination with hydrophobic chromophores (F7,Fll-F14), permethylation and racetylation (F15-F20),and MALD of either free oligosaccfiides (E35,F21-FZ3)or a suitable derivative (F23).MALD shows promise for high sensitivity in the low picomole range. Of course, reductive amination has the advantage of leaving the remainder of the oligosaccharide free, such that sequential endoglycosidase treatment may be followed by stepwise mass spectrometric analysis (F11,Fa). Traditionally chemically or enzymically liberated oligosaccharides were reduced with radiolabeled reagent to the corresponding oligoglycoalditols (F25). Recently, several laboratories have explored a strategy for determination of the linkages by oxidation of the vicinal glycol moieties of the oligoglycoalditols with eriodic acid followed by reduction, permethylation, and LSphlS analysis (F26F33).In addition anomeric configurations may be assigned by com arison of LSIMS spectra of the perdeuterioacetyl derivatives kfore and after oligosaccharide chromium trioxide oxidation (F34). A further breakthrough, particularly for the global characterization of protein glycosylation, has resulted from the use of the new electrospray method of ionization, either on the isolated glycopeptides, peptidoglycans, glycans, or in some cases, the intact glyco rotein. In the case of glyco eptides FZO),information on the Ketero and peptidoglycane (I& eneity of oli osaccharides attached to specific sites is readify obtained, ei&er in the batch mode of the isolated glycopeptide or in the LC/electrospray MS mode directly on a protein est (E!X-E%). Detection of a sialylated branch on the phosp oinositol glycan of the scrapie glycoprotein was readily achieved b electrospray, and subsequently confirmed Interesting resulta on the by high-ener C& analysis (EL?). underivatizfglycosylphosphotidylinositol anchor from the membrane form variant of the surface lycoprotein of Trypanosoma bruceii by negative ion MILD established the microheterogeneity present in the anchor moiety obtained
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after pronase digestion of the protein (F35). While there is little documentation in the literature as yet, initialresults indicate that MALD is able to measure molecular weights of mixtures of oligosaccharides at relatively low levels (picomole range) (E35,F21). Permethylated derivatives give useful fragmentation preferentially at N-acetylhexosamine glycosidic positions, from which non-reducing terminal structural information may be readily obtained. This is of particular interest in location of antigenic determinants (F36) and a variety of other structurally important information (F15).The reductive amination derivatives with octyl paminobenzoate ’eld good ne ative ion fragmentation characteristics by LGMS from whch structural and branching information may be obtained (FlO-F12). However, many times the lower mass fragments of the spectrum are obscured by chemical noise from the liquid matrix. There are also incidents when so-called internal fragments or “double cleavages” appear to be present in liquid SIMS spectra. Fortunately, high-energy CID analysis in the ne ative ion mode of the paminobenzoate derivative eliminates t%mionic s cies from the spectra, while simultaneously removing the cKmical noise so that the lower mass ions may be interpreted unambiguously (F6,F7). High-energy CID analysis has also come into its own by induction of remote-site fragmentation and ring cleavages important in determinjng the structure and branching of the glycoinositolphospholipd anchor lycan from the scrapie prion protein (F18,F19)and glycosptingolipids (F37,F38). The PI lycan structures usually contain a free glucosamine a t t a c h J t o inositol and hence, in this case,there is an advantage to permethylation of such oligosaccharides. The glucosamine forms a positive quaternary center, inducing lv5Xtype fragmentation which is very useful for establishing the branching pattern and location of residues responsible for the microheterogeneity (F18,F19). High-energy CID analysis has also been effectively employed in continuing structural studies of glycosphingolipids (F37)and glycophosphosphingolipids (F38, F39). Structural investigation of Gram-negative pathogenic bacteria has continued, taking advantage of derivatization by a hydrophobic chromophore as the hydrazinobenzoate (F40)in studies of Neisseria gonorrhoeae glycolipid antigens, and those from Haemophilus influenzae (F41,F42). An interesting recent contribution concerns the qualitative CID spectral differences between product and parent ion scan modes (F43).While the product scan spectra were devoid of ring cleavage processes as has been previously reported, the parent scan spectra contain ions which were similar to those reported by Costello and Domon from high-energy CID spectra (F37). Finally, high-energy CID analysis has been shown to provide considerable information on the structure of serine and threonine 0-linked glycosylation from the high-ener CID case spectra of the intact lycopeptides (F44,F45). In of mannosylation an recombinant @-chainhomodimer of platelet-derived growth factor, the site of 0-glycosylation as well as the monomeric or dimeric nature was established (F44), while in the case of fetuin 0-linked glycopeptides, the use of the hydrophobic tBoc-tyrosine derivatives provided e n h a n d sputtering efficiency and sensitivity (F45). 1. Oligosaccharides. In a recent account of research, Albersheim and co-workers discuss what is known about oligosaccharins, carbohydrate molecules that plants utilize to regulate important physiological processes such as plant owth, organ0 enesis, and defense against pathogens (F46). Esing both N d R and liquid SIMS, subunits of xyloglucans isolated from tamarind seed and rape seed hulls were studied and identified as their oligoglycosylalditols (F47).In a very interesting stud , the nature of an alfalfa-specific signal, NodRm-1, was iLntified by liquid SIMS, NMR, % labeling, and chemical modification studies. NodRm-1 was shown to be a sulfated @1,4-tetrasaccharideof D- lucosamine in which three amino groups were acetylated an%one acylated with a C16bis-unsaturated fatty acid (F48).This purified Nod signal specifically elicited root hair deformation on the homolo ous host when added in nanomolar concentration. Another %od factor, Ac-NodRm-1, was urified and identified as an acetylated NodRm-1 on the 8-6of the nonreducing end sugar (F49). Studies of the 0-linked carbohydrate chains in the cellulosome of different Clostridium thermoceltum strains were carried out as the oligosaccharidealditols by NMR and liquid
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SIMS (F50).The complete structure of the exopolysaccharide of Pseudomonas solanacearum strain GMI lo00 was determined by NMR and liquid SIMS and provides the first precise basis for the analysis of the correlation of such structure with pathogenicity in this growth-@ducingphytopathogen of major economic importance in agriculture (F51). A recent paper is devoted to an attem t to answer a long-standin question about the nature of tEe covalent link between araginogalactan proper and peptidoglycan, a key linkage required for the structural integrity of the mycolylarabinogalactan- eptidoglycan-protein complex of mycobacterium, thouggt to be one of the lar est multifunctional polymers within Prokaryotae (F52).T i e strategy pursued in this study depended heavily on the s ecificity of a variety of enzymic and chemical cleavages anfanalysis of the degradation components using methods of NMR and mass spectrometry. Continuation of work on the insoluble matrix of the mycobacterial cell wall has led to the location of mycolic ester substituents (F53). The chemical structure of the hosphomannan of the cell wall of Candida albicam NIH b-’792 (serotype B) strain has been re orted (F54).In connection with the occurrence of pyruvy groups in many bacterid polysaccharides, results concerning the stability of the acetal group and the GC/MS characterization of pyruvic acid acetal substituents present in six polysaccharides with permethylation, methanolysis, and ermethylation reductive cleavage have been reported (F557. An interestin paper on the identification of allergenically active oligosacc! l aritols isolated from sea s uirt H-antigen which claims characterization by liquid SIgS and a variety of other methods, including NMR, was ironically devoid of mass spectral information (3’56).The sea squirt allergy is an asthmatic disease associated with occu ational hazards on oyster farms of Hiroshima Bay, Japan &56). Liquid SIMS has been involved in characterization of the products of oxidative reductive depolymerization of hyaluronic acid (F57). GC-MS of permethylated hexasaccharides established the transient expression of T pe 2 chain in A-active hexa1 cosylceramide of rat s d i n t e s t i n e at weaning time (F58). bggosaccharides from glycoproteins bound to Dolichos biflorus agglutinin isolated from the small intestine of 129 Sv mice were released by endo-&galactosidase and identifie by liquid SIMS and NMR (F59).Sialyl-cr(2-6)-mannosyl-8(14)-N-acetylglymsaminehas been identified in urine of patients with @-mannosidosis(3’60). Using chemical ionization mass spectrometry, methylation analysis, and chromium trioxide oxidation, to ether with NMR, the structures of two oli osaccharides kom the urine of a patient with type 3 gangliosidosis was determined (F61). A variety of studies on oli osaccharides from human milk have been reported (F62-#65). Characterization of both complex and hybridsulfated Oligosaccharides from ovine lutropin has been ac ieved using negative ion li uid SIMS of the methylated derivatives (F66).Liquid SIMI was used to identify the fl-Dxyloside-inducedglycosaminoglycansand oligosaccharidesfrom cultured human skin fibroblasts (F67). The structure and purity of eight new commercial he arm/ heparin sulfate disaccharide standards was confirme using capillary zone electrophoresis, liquid SIMS and NMR (3’68). An extensive report of minor tetra- to heptasaccharides 0-linked to human meconium glycoproteins has been carried out by TLC-MS of neoglycolipid derivatives (F69).A similar study of 22 neutral 0-linked oligosaccharides isolated from bovine submaxihy gland mucin glycoprotein has been carried out (F70).0-Linked fucose was identified in the first epidermal growth factor domain of factor XII, but not found in protein C(F71). The major 0-glycans of the nematode glycoproteins of Toxocara excretoryaecretory antigens were identified as 0-methylated trisaccharides (F72).Liquid SIMS analysis of products of the in vitro incubation of M-GlcNAc and ManaGlcNAc with a(ld)-mannosyltransferaseshows that this enzyme catalyzes the addition of mannose, and initiates outer chain formation in Saccharomyces cereuisiae to the CY(1-3)-mannose residue (F73). This data provides additional evidence to support the revised structure of yeast mannoproteins proposed by Hernandez et al. (FIO). The lyco ptide containing the monosaccharide fucose has been ickntigd in the growth factor domain of urinary-type plasminogen activator (F74). Work on structural characterization of the carbohydrate chains of the recombinant human
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interleukin-4 expressed in CHO cells has been reported (F75). Further characterization of oligosaccharides from CHO cells involves the N-linked structures on soluble CD4 glycoprotein (F76, F77). Carbohydrate structures of human tissue plasminogen activator variant expressed in recombinant CHO cells has been studied (F78).Mass spectrometry was employed in structural fingerprinting of drosophila recombinant tissue plasminogen activator (F79). High mannose-type oligosaccharides were characterized on thyroglobulin in fisher rat thyroid line-5 cells (F80).Using trichloromethane sulfonic acid and liquid SIMS analysis, a sequence and glycosylation site identity of two distinct gl coforms of nonspecific crossreacting antigen was establisied (F81). Using plasma deso tion mass spectrometry and l-and 2-dimensional proton an7l3C NMR spectroscopy, the structure of a streptococcal adhesin carboh drate rece tor on Streptococcus sanguis H1 has been estabfshed (F827 2. Glycolipids. As not’ed in the previous review, mass s ctrometry has played a predominant role in the structural cGacterization of glyms hingolipida for more than 2 decades by major groups in Sweien, the United States, Japan, and Germany. Initially concerning elucidation of the nature of and more blood oup antigens (F83), tumor antigens (F84), recentr as receptors for bacteria responsible for adhesion, robab$ many of these same structures on glycoproteins will l e shown to play a major role in immune system function. Current studies address the identification and composition of glymsphingolipidsin marine organisms, parasites, and other invertebrates, as well as continuin studies on mammalian systems. Studies of gan6liosides kom the eggs of the sea urchin, Anthocidaris crass1spinu, reveal great contrast to their previously reported analogs in sperm, in that they are more hydroxylated in the sialic acid, fatty acid, and long-chain base (F86).The occurrence of ceramide digalactmide in the marine spon e, Halichondria japonica, has been characterized (F87). Worf on the ganglioside from the echinoderm Acanthaster planci, a marine invertebrate, has been presented (F88). Investigation of the lycosphingolipids from the liver of the rainbow trout, Oncortynchus mykiss, revealed a surprisingly high concentration of 9-0-acetyl-GD,, scarce in mammals but abundant in human melanoma. It was sug ested that trout liver may be a source of this antigen (F897. Several novel fucose-containing glycosphingolipid immunogens have been isolated and characterized from the eggs of the parasitic trematode, Schistosoma mansoni (F90).A detailed study of fractions containing tri- to pen lycos lceramides isolated from the green fresh water algaeyhloreia kessleri, has been reported (F91). Recently, the presence of an additional group of glycosphingolipids from the pupae of Calliphora uicina (Insecta: Diptera) which are amphoteric in property, containin Nacetylglucosamine-linked phosphoethanolamine has Eeen reported (F92). The presence of disialosyl-lacto-N-neotetroeylceramide has been reported in adult mouse cerebellum (F93). Identification of a hexaglycosylceramide in the extract of liver from a human transplant patient showed that it belon ed to a blood group Leb (F94).Using high-energy CID * yl-GM1 anglioaides have been isolated and anJysis la c h a r a c t e r i z m rat spfeen (F95).The separation of extremely minor gangliosides G and $la was carried out for the production of monoclonJlabntibodies, which were subsequently used for the determination of developmental expression of GMlb in chick brain (F96).Isolation and characterization of a novel A-active octaglycos lceramide with a type 1chain repeat has been established, mishown to be inherited as a recessive trait in epithelial cells of the small intestine of inbred rats (F97).A detailed structural analysis of the YAC-1 gangliosides demonstrates that the gangliosides of the G pathway IVNeuAc/NeuGc-GgOse,Cer (GM1 and IV&! uAc/NeuGc-GgOse Cer (GdNAC-GMlb) were the main gangliosides expressed tF98). Mass spectrometry has been used in conjunction with degradation with exoglycosidases and immunostainin techniques for the screening of myeloid monoclonal a n t i d i e s for specificity of recognition of carbohydrate structures (F99). Similar work was carried out for the characterization of T cell antibodies and their recognition of gangliosides (F100). Studies of the lung colonization potential of in vivo selected sublines from mouse L1-fibrosarcoma cells showed these sublines to be associated with the dramatic acceleration of
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polar ganglioside synthesis, particularly GDla(F101).Mass s ectral and NMR analyses revealed that a preparation of gPobotriaosyylceramidefrom human kidney contains two major molecular species comi ating (F102). Using a monoclonal antibody, 188C1 raisefagainst skin tissue on the back of bullfrogs (Rana catesbeiana), a novel common antigen in neural and intestinal tissues of chicken was shown to be a novel hybrid structure of type 2 Le’ epitope and GM an lioside core structure, designated as Lex-GMl(3’103). has been used to characterize unmodified endotoxin preparations constituted of Re-, Rd-, and Rc-type lipopolysaccharides from the Salmonella minnesota 595 species of enterobacteria (F104). The L4 reactive antigen from M. tuberculosis, L4-PIM, was isolated and shown to be common m cobacterial dimannosylated phosphatidylinositol PIMz ( 4 0 5 1 . PDMS was used to screen structural differences between two components, usually present in unequal quantities in Bordetella pertussis endotoxin preparations (F106). A series of monophosphoryl Lipid A homologs obtained from Salmonella minnesota Re595 lipopolysaccharide have been characterized by liquid SIMS (F107). High-energy CID analysis has been important in establishing the structural nature and heterogeneity of the Haemophihs drucreyi Gram-negative human mucosyl pathogen that causes canckroid or genital ulcer disease (F108). Further studies by hi h energy CID analysis established the structure of inositol sp!&ophospholipids from cotyledonsof yo peppers which rot.& them against necrotic lesions i n d u d y y the athogen Shytophthora capsici (F109). However, work on cKaracterization of the oligosaccharide epitopes of Neisseria meningitidis have been reported (F110).The structure of Leishmania mujor li hosphoglycan has been investi ated by 2-dimenand negative ion liquid SIMb (F111). sional Recently, characterization of a recombinant glycolipid by liquid SIMS established that this species contains the lipopeptide core originating from Mycobacterium smegmatis, whereas the oligosaccharide se ment arose from the cloned Mycobacterium avium genes fF112). This extremely important work is the first demonstration of the cloning and expression in nonpatho enic mycobacterium of the genes encoding complex cell w& lycocon’ugatesfrom a pathogenic mycobacterium. This bre& hroug presents a new strategy for investigating the role of such glycolipids in disease processes (F112). G. FOREIGN COMPOUND METABOLISM AND PHARMACOKINETICS Applications of mass spectrometry to studies on the fate of xenobiotica in living systems continue to expand at a rapid pace, in keepin with the trend noted in the previous review in this series &I). In large part, advances in this area of ap lication reflect technical developments in ionization tecfmques and on-line chromatographic-mass spectrometric couplings for the analysis of trace uantities of polar metabolites in highly complex biologicalrhuids,and there is little doubt that mass spectrometry now is accepted as an indisensable technique in studies of foreign compound metabom. Of particular importance in the past 2-year period have been developments in electrospray and related atmospheric pressure ionization techniques and their commercial adaptation for use in LC-MS instruments equipped with either sin le or tandem mass analyzers. Recent pertinent articles antreviews have dealt with electrospray ionization (122, G3), of API mass spectrometry (G4, G5) including LC-MS ? ? 7 ) , tand em mass spectrometry (G8, G9),thermospra LC-MS (GIO),and continuous-flow FAB-MS (GI1). In adi dition, a cha ter has a peared on the use of mass spectrometry in pharmacoyogy (G12r and applications of maw spectrometry to the fields of drug metabolism and toxicology during the period 1988-91 have been reviewed (G13). An increasingly popular approach to the identification of drug metabolites is to employ soft ionization methods to analyze fractions obtained from HPLC separations of biological fluids. Exam les where FAB-MS has been used in this manner inclufe studies on the biotransformation of the HMG-CoA reductase inhibitors lovostatin (G14) and pravastatin (G15),the pharmacologically-activepe tide derivatives cyclosporin A (C16-G18) and RS-26306 ((?IS), the histamine H2 antagonist etintidine (G20),and the antiinflammatory agent diflunisal which is converted to an unstable
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hydroxylated product (G21). There has been much interest in the antitumor properties of taxol, a tetracyclic diter ne, and both FAB-MS and desorption CI-MS have been empcyed to identify metabolites isolated from rat bile (G22). FAB-MS was used to demonstrate that codeine is metabolized to reactive a,&unsaturated products which form adducts with sulfur nucleophiles ( G B ) ,while desorption CI techniques were employed to characterize metabolites of drugs such as tiospirone ((724, G25), methapyriline (G26), and zonisamide (G27). The latter report is of interest in that thermospray ionization was used in addition to CI, althou h in an off-line fashion as opposed to the more conventionafLC-MS mode. A novel type of drug metabolite was identified in a study of the fate of valproic acid in rat liver mitochondrial preparations; isolation of a polar product by HPLC and analysis by Cs+ ion LSIMS/MS revealed that the compound was an acyl adenylate derivative of the parent drug (G28). Presumably, this mixed anhydride species was formed as an intermediate in the conversion of valproate to its coenzyme A thioester conjugate. For some years, reversed-phase HPLC has been the primary chromatographic technique used in drug metabolism studies, replacing GC methods. Not surprisingly, LC-MS techniques have become relatively more important as a result, and thermospray has hitherto been the most commonly-used interface for LC-MS couplings. Examples of metabolic studies based on TSP LC-MS include work on the esticide methoxychlor (C29), the experimental acyl-8oAcholesterol transferase inhibitor CI-976 (G30),the antidepressant doxepin (G31), the antihistamine terfenadine (G32), and the thiocarbamate herbicide sultan ((333). Biotransformation of arteether, an antimalarial natural roduct with a labile endoperoxide structure, has been stu&ed by TSP LC-MS (G34), which also has been employed to identify glucoside conj ates of carbovir,a carbocyclic nucleoside (G35),and p h e n o d i t a l , an antiepileptic barbiturate (G36). In the case of another barbiturate, heptobarbital, TSP with tandem mass analysis was emplo ed to identify a novel unsaturated metabolite of the drug (d37). A number of examples have appeared of the use of CF-FAB-MS for metabolic studies, such as those dealing with sulfon 1urea herbicides (G38) and various peptide analogs (G39, &O). Although the particle beam interface has been used in LC-MS studies of drug metabolism (G41(3431, this type of coupling appears to have found greater utility in work with nonvolatile compounds of environmental, rather than pharmaceutical, interest (G44). In contrast, the introduction of API sources for LC-MS instruments (particularly with tandem maw spectrometers)is beginning to have a dramatic impact on the field of drug metabolism and pharmacokinetics, in that an impressive range of compounds of high to intermediate polarity (re ardless of molecular weight) are bein found to be amenatle to analysis by this approach (G4-Gfi. Current indications are that API systems equipped with electrospray, ionspray or heated nebulizer interfaces provide very hi h sensitivit of detection, and that than response fadors are mud! less dependkt on sample has been the experience with TSP. Moreover, t h e a f f e d n in the interface are minimized with such API instruments, which have been applied recently to the stud of a variety of thermally-sensitive metabolites (C45) and drrug conjugates (G46-G53). The successful couplin of API sources to microbore (G54) and packed-capillary ( 8 5 5 )LC columns would ap ear to hold great promise for the routine application of L&MS to many areas of biomedical research. Finally, it may be noted that LSIMS techniques have been employed in concert with thin-layer chromatography to obtain spectra of nonvolatile or thermally-labile drug metabolites by scanning TLC plates (G56). During the past 2 years, there has been a striking increase in the frequency of metabolism studies which have taken advantage of tandem mass spectrometry (G8, G9). Many investigators have employed MS/MS to “screen” complex biological samples for the resence of drug-related materials which often are revealed gy the generation of structurallydiagnostic fragment ions or neutral losses resulting from cobional activation of mass-selected parents. Once detected in this fashion, individual metabolites then may be identified on the basis of their individual product ion spectra. Examples of this powerful approach to ‘metabolic profiling” include studies on the biotransformation in mice of the antischistoANALYTICAL CHEMISTRY, VOL. 04, NO. 12, JUNE 15, 1992
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s o d drug praziquantel, of which no less than 17 h dro lated derivatives were detected in urine extracts by SI-M? MS (G57, G58);the identification in monkey urine of oxidlzedand conjugated metabolites of an experimental antianxiety drug, U-78,875,by FAB-MS/MS (G59);the excretion of metabolites of pyrilamine in rat urine by TSP LC-MS/MS (G60);the presence of S-oxidized derivatives of a new calcium channel entry blocker, AJ-2615,in rat plasma (G61);and the formation of metabolites of mifentidine (G62)and cimetropium bromide (1363) b he atic microsomal preparations in vitro by FABM S / M ~ egant work on the biotransformation in rats of omeprazole, a otent inhibitor of gastric acid secretion, has Weidolf and Coyey (G48) who employed been re orted L,C-APp/MS f for detection of biotransformation products. This study is noteworthy in that the authors employed the isotope cluster technique (using the %%labeled analog of omeprazole) in con'unction with MSjMS to detect ,and identify over 40 metaLlites of the drug m a partiallypunfed urine extract. MS/MS also has proven to be a powerful method for confirmin the presence of a known metabolite in a biological fluid. approach is useful in do ing control applications, an exam le of which would be the etection in horse urine of detomi ine carboxylic acid, the ma'or e uine metabolite of the analgesic drug detomidine ( d 6 4 , B65). Recently, GC-CI/MS/MS on an ion trap mass spectrometer has been em loyed to characterize metabolites of 3,4-(methylenedioxyyamphetamine in the rat, when evidence was obtained for the formation of hydroxylated ring-opened products which are potent neurotoxins (G66, (367). One of the benefits of soft ionization MS methodology for drug metabo 'sm studies is that polar conju ates (e.g. with glucuronic or sulfuric acid) may be analyzefas the intact species. While the classical approach, involving hydro1 sis of the conjugate to the correspondin a lycone followe by derivatization and MS analysis, still tfntfs some application, it is indirect in nature and therefore is inherently problematic. Moreover, several classes of drug conjugates are refractory to hydrolytic cleavage althou h, in favorable cases, conjugates may be derivatized and aniyzed b EIMS (G68). Currently, drug conjugates involves the preferred strategy for work FAB-MS investigation of HPLC isolates, or on-line LC-MS analysis of biological fluids. Glucuronide conju ates are well-suited to this approach, and numerous exampyes of the identification of ether-, ester-, N-linked and quaternary ammonium glucuronides are to be found in the recent literature ( G 6 9 4 8 9 ) . FAB-MS a pears to be the technique of choice for such work, probably L u s e TSP LC-MS does not always afford prominent MH+ or [M - HI- ions from glucuronides and sulfates (G78). Judicious choice of derivatization procedures often can be employed to distinguish between regioisomeric glucuronides (or other types of adduct) where conjugation may occur at more than one center (G75). Characterization of intact glucuronides is critical in the case of amines because in many instances the adducts actually derive from conjugation of the carbamic acids to which the parent amines are transformed in biological media (G90-G93). Attempts to hydrolyze such carbamoyl glucuronides rior to isolation will result in decarboxylation of the unstab e carbamic acid and recovery of the free amine, and thus the adduct would be mis-identified as an N-glucuronide. Conjugation with coenzyme A represents a potentially important athway for the metabolism of carboxyhc acids, and FAB-MS been employed successfully to characterize intact coenzyme A thioesters of valproic acid (G94), flurbiprofen (G95) and ibuprofen (G96). Conjugates with the tripeptide glutathione, which are of great toxicological interest, also are amenable to study by several soft ionization techniques and are discussed below (see section H, Toxicology). Despite the prominent role of soft ionization methods in contemporary studies of foreign compound metabolism, conventional E1 and CI techniques remain extremely valuable for structure elucidation in those situations where the metabolite is sufficiently stable thermally (or can be rendered so by ap ropriate derivatization) to be analyzed by GC-MS. Althoug! the recent literature contains many such examples, thoee dealing with the identification of metabolites of the novel antiepileptic agent stiripentol (G97),the cannabinoid derivatives cannabidiol (C98) and cannabichromene (G99),the methadone analog recipavrin (ClOO),and the antidepressant trimipramine (GlOl) serve to illustrate the high sensitivity,
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versatility and structural information available from 'classical" GC-EIMS methodology. Indeed, a novel pathway of biotransformation, "N-depyridination", was detected during a study by GC-EIMS of the metabolism of tri lennamine, an . an an&, metabolic antihistamine ( ~ 1 0 2 )[Interestingly, reaction involvin cleavage of an aromatic ether was detected recently by TSP fC-MS (C103).] However, it should be borne in mind that derivative formation occasionally can lead to artifact formation, as occurred when metabolites of valproic acid were treated with hydroxylamine and subsequently trimethylsilylated (G104). The use of stable isotopes now is commonplace in studies of drug metabolism, and a number of algorithms have been developed for the deconvolution of mass spectral peak abundance data from experiments with stable-isotope-labeled compounds (G105-Gl07). The isotope cluster technique is particularly widely used in xenobiotic metabolism work since it can greatly facilitate the recognition of drug-related materials in complex biological extracts (G48, Cl08). The heightened awareness of stereochemical issues in the dis 0sition of individual isomers of chiral drugs has led to stu ies with seudoracemic mixtures, exemplified b investi ations on gaiopamil, a calcium-channelantagonist 6 1 0 9 ) . kterest in mechanistic aspects of biotransformation processes has prompted several investigations with substrates labeled at specific sites with deuterium (Gll&G122), while metabolic oxidation reactions may be studied profitably in vitro by the use of lSOas a tracer (G123-GI25). In a recent report, Meese et al. (G126) used GC-EIMS and high resolution EIMS together with a 13C-labeled variant of the mucolytic agent carbocysteineto demonstrate that the major metabolite of this compound in human urine was S-(carboxymethy1thio)-Lcysteine, and not carbocysteine S-oxide as was believed previously. This is an important finding because biotransformation of carbocysteineto the metabohte in question has been s gested to exhibit genetic polymorphism, and the respons 8 e pathway therefore has attracted much attention. Clearly, it is imperative that the end-product metabolite of the enzyme involved in the potential olymorphismbe identified correctly. Stable isotopes also are%eing applied more widely to harmacokinetic investigations, and the relative merits ant! limitations of such tracer techni ues have been reviewed by Browne (G127). Asp& of thelisposition of hen been examined with the aid of a doubly-la ele (13C,15N) malo of the drug, which circumvented problems associated with &e nonlinear pharmacokinetics of this antiepilepticagent (G128-Gl30). The pharmacokinetics of nicotine in man have been examined by means of 3',3'-dideuteronicotine (G131, G132), and the disposition of the individual enantiomers of racemic meto rolol (G133), felodipine (G134), and hexobarbital ( G l d have been studied with the aid of pseudoracemic mixtures. Most investigators continue to favor the use of stable isotope labeled internal standards for quantitative mass spectrometric procedures, and although deuterium is by far the most commonl employed isotope for this purpose, the heavy isotopes of cargon, nitrogen, and oxygen are ado ted occasionally for stable isotope dilution assay. Thus, Dogson analog as inet al. (G136) used a multiply-labeled (13C, temal standard for tebufelone, a novel a n t i i d h u mtory drug. This paper is of interest because the method was based on selected reaction monitoring GC-EIMS/MS, and the work serves to demonstrate that highly selective and sensitive MSf MS assays performed on triple quadrupole instruments also can be robust, since over 3000 plasma specimens were analyzed successfully for tebufelone over a 2-year period. A related report on the determination of indomethacin in human plasma down to levels of 0.1 n mL-' also employed selected reaction monitoring G C - M S / b , although negative ion CI ionization was employed here (G137). A different type of application of stable isoto es to metabolic studies is found in the 13Cbreath test, in w k h the N-demethylation of s ecifically-labeled probes, such as [13C]caffeine and [ 1 3 E ] aminopyrine, is studied in vivo. The exhaled W02resultin from this reaction is measured by isotope ratio MS, and provides a noninvasive method to examine the activity of selected cytochrome P-450 isoenzymes (G138). Abramson and colleagues have continued to pioneer the development of the chemical reaction interface-mass spectrometry (CRIMS) approach to the detection of compounds labeled with stable (G139-6'142) and radioactive isotopes ((3143). Earlier work
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on GCqRIMS now has been su lemented by studies on the of the need for sensitive methods for the analysis of specific cou ling of LC columns to the CflpMS interface (G141,G142), chemical warfare a ents and their mammalian metabolites. ancfthe resultin isotope-selective LC-MS detection systems Ketkar et al. (H5)ave reported on the development of an API MS MS method for the detection, in real time, of the should prove to %e highly valuable for use in drug metabolism chemic warfare agents sarin (GB) and VX in ambient air studies with isotopically-labeled tracers. The combination of high sensitivity and high selectivity of at concentrations below 20 ppt, while Black and co-workers detection are always key considerations in MS-based assay at the UK Chemical Defence Establishment have focused on the use of GC-CI/MS methodology for the detection of urprocedures and it is not surprising, therefore, that the negative ion CI approaches which have proven so successful in the inary metabolites of sulfur mustard in potential victims of analysis of eicosanoids and bio enic amines have been exexposure to this toxic agent (H6, H7).Workers at the US Army Research Institute of Chemical Defense have reported tended to the determination of row levels of drugs and their metabolites in biological fluids. Examples of the use of GCon a GC-MS approach for the detection of the organo"IC1 MS for pharmacokinetic investigations published during phosphonic acid derivatives of soman (GD), sarin, and GF the present re orting period have included studies on clenwhich are present in blood and urine following exposure to Although not presently classified buterol (G143K flecainide (GI&), 3-amino-1-phenylbutane the parent nerve gases (HS). as a chemical warfare agent, the marine toxin saxitoxin is (a chiral metabolite of labetalol) (GI#), naltrexone (G146), terbutaline (G147),indomethacin (G137), LSD ( G I B ) ,and regarded as a candidate for such use, and efforts have been valproic acid (G149). Interestingly, in a recent Technical Note, directed toward the development of methods to confirm the Abdel-Baky and Giese (G150) reported that optimization of presence of this compound at trace levels. The most promising parameters for negative ion detection on a commercial a roach to date ap ears to be a method based on ionspray uadrupole GC-MS system led to detection limits for selected I $ ! which was deveiped recently to monitor levels of saxitoxin in s h e w flow ijection analysis,LC-MS, and capillary Buorinated analytes in the zeptomole mol) range. This electrophoresis-MS were compared as sam le introduction report emphasizes the exceptional sensitivity theoretically techniques, and each proved to be a viabg method (H9). attainable with NI CI-MS of suitable electrophoric compounds. In recent years, the field of biochemical toxicology has Additional applications of quantitative MS assays have been continued to expand rapidly, and mass spectrometry has to the simultaneous determination of cocaine and its major played an essential role in many types of studies dealing with metabolites in human hair as a means of detecting cocaine the molecular basis of forei compound-induced toxicities. abuse ( C l S l ) , and to the separate measurement of the 6Chemicallyreactive metaborbs are known to be responsible lactone and hydroxyacid forms of the HMG-CoA reductase for the adverse effects of many compounds, and the identiinhibitor simvastatin in human plasma using the cyclic ferfication of these short-lived species and the characterization roceneboronate derivative (G152). An unstable N-acetylof their covalent adducts to cellular macromolecules have cysteine conjugate of the toxin methyl isocyanate has been become bur eoning areas of research. Free radical intermeuantified in rat urine by means of a stable isotope dilution diates, whicfi are formed during the biotransformation of a %SPLC-MS assay (G153),and several reports have a variety of classes of chemicals, cause cellular damage by recently on the application of LC-API MS (and li&%$mechanisms which include lipid peroxidation. In some cases, techniques to the quantitative determination of drugs and these radicals can be spin-trapped and LC-MS techniques their metabolites in biological fluids (G154-GI57). Based on (TSP and electrospray) have been used to identify the the favorable results from these latter studies in terms of roducts resulting from fatty acid (H10) and alkyl radicals sensitivity, linearity of response, accuracy, precision, and ease H11).An alternative (indirect) approach to the study of free of use, it may be anticipated that LC-MS systems equipped radicals is to employ GC-MS methods to analyze specific with electrospray or ionspray sources will be employed with products of fatty acid peroxidation. Recently, a sensitive increas' fr uen in the future for routine harmacokinetic procedure has been reported for the determination of unalso may be employexfor quantitative studies?FyAB/xI saturated hydroxy fatty acids present in tissue, which involves work, as has been demonstrated for the antibiotic prodrug the extraction, reduction, and trimethylsilylation of these erythromycin 2'-ethylsuccinate (G158). An exciting devellipids, followed by analysis by NI CIMS (H12).Stable opment in the application of MS to pharmacokinetic studies products formed by radical-coupling processes often reveal has been the coupling of microdialysis probes (GI591 to mms the identities of the radicals themselves, and MS has been spectrometers, which offers the prospect of monitoring conused to identify the products formed during eroxidative centrations of drugs, their metabolites or endogenous subreactions involving aromatic amines (H13, H147. Man restances in hysiological fluids effectively in real-time. Caprioli active intermediates are even-electron electrophiles, anJMS and Lin (&So) described an MS/MS method for determinii has been em lo ed to probe the mechanism of their formation blood levels of penicillin G in a rat which had been equippe! with the aiaofauxiliary techniques such as stable isoto e with a microdialysis probe surgically im lanted in the carotid labeling. Metabolic activation of bromobenzene, a m o g l vein. The effluent from the robe (4.8 gE, rmn ) was collected hepatotoxin, leads to the generation of reactive intermediates in a loop in'ector and passe# at suitable intervals to the CFwhich bind covalently to protein and form conjugates with FAB interflace of a tri le quadrupole mass spectrometer. sulfur nucleophiles. Mass spectrometric studies on these Off-line microdialysis-hS approaches also have been emadducts, and the corresponding adducts from [2H4]bromoloyed in pharmacokinetic studies. Thus, Menacherry and benzene, has provided an insiiht into the role of reactive arene fustice (G161)examined the kinetics of the e rimental drug oxide and quinone metabolites in the toxicity of this comGBR-12909 in rat brain tissue by using TSPyC-MS/MS to H16). Bromobenzene also is metabolized to pound (H15, quantify drug levels in dialysis fluid, while C et aL (G162) 4-bromocatechol, and the mechanism of this process in vitro employed a stable isotope dilution GC-EIM approach to has been investigated using [p-?H]bromobenzeneas substrate assess the influence of amphetamine administration to rats (H17). Interestingly, the results of this study demonstrated on cerebral biogenic amine levels using brain microdialysate that the pathway followed de ends upon the in vitro prepafluid. It may be anticipated that these emerging techniques, ration used. Mechanistic stu8es on the metabolic activation together with the use of ion tra s for the qualitative and of acetaminophen in vivo were conducted with both '80- and uantitative determination of {rugs in biological fluids 2H-labeled analogs of the drug, and sulfur-containin me$~GISGI~B), will play an increasingly important role in future tabolites in excreta were converted to a novel volatile ierivstudies of foreign compound metabolism and pharmacokiative and analyzed for heavy isotope content by GC-E1 MS netics. (H18).Based u on the isotopic enrichments of the acetaminophen metdolites, it was possible to construct an inteH. TOXICOLOGY grated reaction scheme which accounted for the formation of all known oxidative metabolites of acetaminophen from an Applications of mass s ectrometry to the analysis of eninitial oxygen-centered free radical species. The potent nigand sticide residues (H2) have vironmental pollutants roatriatial toxin MPTP has been the subject of much interest been surveyed durin the eriogovered by this review, as since this tetrahydropyridine derivative requires metabolic have applications in t%e nerd of forensic toxicology (H3,H4). oxidation to the corresponding pyridinium compound for the Consequently, these topics will not be dealt with again here. expression of its adverse effects. Deuterium isotope effects The recent roliferation and use of chemical weapons, parhave afforded valuable information with regard to the ticularly in t1e Middle East, has led to an increased awareneas
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mechanism of this oxidation rocess (H29).Recent1 , the findings from these studies on bPTP have been extenied to the clinically important anti ychotic agent haloperidol, which was found to undergo metarolism in rats to a polar product. This metabolite was identified by LC-MS as the pyridinium derivative of haloperidol, which was shown to deplete do amine in stiatial nerve terminals (H20).The putative hydropyridinium intermediate in the overall 6-electron oxidation of haloperidol was synthesized and found to undergo both disproportionation to yield the pyridinium metabolite and intramolecular cyclization to a product which was idenRelated studies on the oxitified by E1 and CI MS (H21). dation of i eridine-containing compounds by cytochrome P-450 mofefsystems have provided further evidence for the role of reactive iminium ions in the corresponding enzymemediated process (H22). An unexpected competing metabolic reaction for the psychoactive piperidine derivative phencyclidine (PCP) has been found to be C-formylation,the product from this reaction being identified lar ely on the basis of its high resolution E1 mass spectrum (&3). Additional examples of chemically-reactive intermediates which have been studied with the aid of MS techniques derive from work on the metabolism of the toxic natural product and halocarbons such as 1,2-dichloromenthofuran (H24), l,2-dibromo-3-chloropropane(H26), tripropane (H25), halovinyl 2-nitrophenyl disulfides (Ha), chloroethylene (H27), and the N-acetylcysteine conjugates of isomeric fluoroethylenes (H29).Ethyl carbamate, an established rodent carcinogen, has been found to undergo two sequential cytochrome P-450-mediated oxidation reactions to yield a highly reactive epoxide which binds to DNA; GC-CI MS was used to identi the intermediate (vinyl carbamate) in the process (H30). F%3 -MS was employed by Eisenbrand and co-workers to identify an N-acetylc steine conjugate of coumarin in rat urine (H31).This fining was taken as evidence for the conversion of coumarin to a reactive 3,4-epoxide intermediate. In the case of PhIP, a mutagen which is fomed in certain meat products durin cooking, metabolism was found to generate a enotoxic N-iydroxy derivative which was identified by F ~ B - M S( ~ 3 2 ) . Conjugation with glutathione (GSH) represents an important route of detoxification for many reactive intermediates, and the structural characterization of GSH adducts affords a means by which the reactive metabolites themselves can be identified. This is most effective1 accomplished by isolation of the conju ates from bile by dPLC, followed by analysis using LSIMEf or FAB-MS. An interesting example of an endogenous compound (as opposed to a xenobiotic) which can undergo metabolism to a reactive intermediate is bilirubin-IXa, the major degradation product of protoheme. Bilirubin normally is converted to a glucuronide conjugate prior to excretion, but in the Gunn rat, which is deficient in this pathway, metabolism occurs at the C-3 and C-18 vinyl groups to yield chemical1 -reactive epoxides. The latter in the formation of four species then react with G S d resul isomeric adducts which have been c aracterized recently by LSIMS (H33, H34).Usually, conjugation with GSH is mediated by GSH transferase enzymes, although in certain cases adducts are formed nonenzymaticall Midha et al. (H35) have used FAB-MS to show that a n u d e r of antipsychotic thioxanthene derivatives are converted to GSH conjugates by a novel non enzyme-dependent process which involves addition of GSH across the exocyclic double bond. Some unusual adducts have been identified during the present reporting period, including a mixed GSH/cysteine conjugate of the and mixed ne hrotoxic com und dichlorovinylcysteine (H36) GlH/glucuronigconjugah of the hepatotoxic terpene pulegone (H37); in the latter work, conjugates were detected by the use of the isotope cluster technique and an MS/MS-based screening strategy which involved the conversion of GSH adducts to N-(benzoyloxycarbonyl)methyl ester derivatives prior to mass spectrometric analysis (Ha). [Rela+ MS MS screening ap roaches for this important claas of conjugate L v e been descrifed by Gaskell et al. (H39).] Further examples of unusual GSH conju ates which have been characterized by MS techniques incfude an adduct to the oxidized food mutagen PhIP (H40), a conjugate of the desacetyl derivative of spironolactone (an antimineralocorticoid) where the linkage is ma a disulfide bond (H411, and a GSH conjugate of methyl isocyanate (which is generated as a metabolite of N-
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methylformamide) where the adduct contains an unstable carbamate thioester linkage (H42, H43).Spontaneous reversal of the conjugation process with isocyanates can occur under raising the possibility that physiological conditions (H4%H44), GSH conjugates of isocyanates may sewe, in part, as transport forms of toxic isocyanates in vivo (H45). A similar phenomenon has been noted recently with isothiocyanates (H46). FAB-MS (using either positive or negative ion detection) has been used to identify GSH conjugates from the followin (H47), butylate8 compounds: 3-tert-butyl-4-hydroxyanisole hydroxytoluene (Ha), benzene (H49), paminophenol (H50), dopamine and a-methyldo amine (H52),bue enol (H51), and dithiopyri&ne (H54). d i e n e monoxide (H53), In an effort to extract structural information from the GSH adduct derived from the antineoplastic dru cyclophosphamide, Fenselau and co-workers (H55) empfoyed a combination of laser desorption and E1 to generate an odd-electron parent ion from the adduct. This M + species underwent site-specific fragmentation, which allowed the authors to perform an experiment with [2H4]cyclophosphamideand thereby gain an insi ht into the mechanism of conjugate formation. collision3activation of even-electron parent ions formed by FAB-MS is a more common approach to obtaining structural information from GSH conj ates, and the same group has re rted on the application o%gh energy (6 keV) of the above collisions to g k n e the sites of deuterium labe [2H4]cyclophosphamide-GSHadduct (H56)YAB-MSlMS has also been used to study GSH conjugates from pule one (~37), various haloalkanes ( ~~3 5 7, ) )spironolactone , &I), isocyanates (H42-H44), naphthalene oxide (H58),the toxic pyrrolizidine alkaloid monocrotaline (H59),and 2-furamide (H39). In contrast to FAB-MS, TSP LC-MS does not always although yield molecular ion species from GSH adducts (H60), postcolumn addition of organic modifiers to the mobile phase Neverthela, TSP has been may alleviate this problem (H62). employed successfully to identify the GSH conjugates of and (methycompounds such as hexafluoropropene (H62) 1enedioxy)methamphetamine (H63),when "class characteristic" fragment ions often are observed at m z 129, 147 and [MH+ - 1291. Much more promising than SP are the emerging LC-API MS techniques, such as ionspray or electrospray with a heated nebulizer, which have been employed to study the GSH adducts of methazolamide, an anti laucoma drug (H50), (E)-2-propyl-2,4- entadienoic acid, a tepatotoxic metabolite of valproic acid &51), 2-furamide, a model hepatotoxin (H52), and mitoxantrone, a novel cardio rotective agent (H53). The API a roach generall afforis abundant MH+ species from GSffconjugates aniexhibits very high sensitivity of detection. As ointed out 1system elsewhere in this review, the ease of operation of b and their compatibility with reverse-phase mobile phases render them ideally suited to the analysis of GSH adducts and related polar drug metabolites. Pesticides often undergo biotransformation via the GSH pathway, and typically give riee to a complex array of polar end-products such as sulfonic free thiols and S-glucosides (H65). It may be acids (H64), anticipated, therefore, that LC-API MS will find great utility in the area of agricultural chemistry. In addition to GSH, peptides, proteins and nucleic acids may be subject to covalent modification by chemically-reactive metabolites, and there has been considerable interest in defining the structures of these adducts. Mass spectrometry has been invaluable in this area of toxicology, particularly in view of the low levels of xenobiotic-macromolecule conjugates which usually are encountered. Studies with amino acids and model peptides can be helpN in predicting the nature of the correspondin adducts to proteins, as exem lified by a study which used !F-FAB/MS and TSP LC-& to identify the reaction roducta formed between a series of N-acetylamino acids anfmetabolites of the nephrotoxic c steine conjugate S-(1,1,2,2-tetrafluoroethyl)-~-cysteine(d36). Similarly, FAB-MS/MS was employed to characterize the adducts to the model peptide oxytocin generated by two reactive conjugates of methyl isocyanate, viz. S-(N-methylcarbamoy1)Ha). cysteine and S-(N-methylcarbamoy1)glutathione(H67, Several enzyme inhibitors are known to inactivate their respective target enzymes by binding covalently to amino acid residues at, or close to, the active site, and elucidation of the resulting adduct structures can provide valuable information on the underlying mechanisms. Examples where MS played
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MASS SPECTROMETRY
a key role in such studies include recent work on the inactivation of 4aminobutyrate aminotransferase by the antiepileptic agent vigabatrin (H69), of dihydropyrimidine dehydrogenase by 5-iodouracil (H70) and of ornithine decarboxylase by a-(difluoromethy1)ornithine(H71). Environmental or occupational exposure of humans to toxic a ents can, in principle, be monitored by the excretion of cLacteristic urinar metabolites, e.g. the carcinogenic polycyclic aromatic hydrrocarbon benzo[a] yrene, a ubiquitous environmental contaminant, is metagolized to phenolic sulphate conjugates and cysteine adducts which can be determined by techniques such as CF-FAB/MS (H72, H73). Similarly, the thiadiazole fungicide etridiazole is metabolized to a carboxylic acid derivative and an N-acetylcysteine conju ate which are excreted in urine and can be assayed by &-EMS methcds (H74). However, u s d y it is not pcasible to monitor long-term exposure to xenobiotics by this approach. For this reason, protein con'u ates in general, and adducts to hemoglobin in particular (wgose lifespan is in the order of 4 months), have become the focus of many dosimetry studies for chemical carcinogens (H75, H76). Thus, hydrolysis of hemoglobin obtained from red blood cells may be em loyed to liberate specific carcinogen-amino acid adducta, whict then can be identified and quantified b GC-MS procedures. Examples of such applications have een to assessments of occupational exposure to ethylene oxide (H77), 4,4'methylenedianiline (H78), acrylamide (H79), and butadiene (H80), and of environmental exposure to polycyclic aromatic Many h drocarbon epoxides and diol e oxides (H81-H85). orthese assay methods employ NYCIMS detection, and thus are highl sensitive and specific. Certain therapeutic agents also binicovalently to hemoglobin, such as acetaminophen which is metabolized to a reactive quinone imine derivative, NAPQI (H86). Cysteine residues are alkylated preferentidy by NAPQI and by the related electrophile 1,4-benzoquinone (H87). Several anticancer l-(2-chloroethyl)-l-nitrosoureas form hydroxyethyl adducts with the N-terminal valine residue of hemoglobin, and its has been proposed that GC-MS assay of N-(hydroxyethy1)valine (as the trimethylsilyl derivative of ita [pentafluorophenyl]thiohydantoin)may represent a suitable means of monitoring exposure of atients to this class of chemotherapeutic alkylating agent 8388). The study of chemical modifications to DNA presents numerous analytical difficulties, not the least of which is that the levels of modified bases in vivo often are extremely low. A sensitive stable isotope dilution GC-MS method has been developed to quantify a product of oxidative DNA dam e, 5-(hydroxymethyl)uracil, which can be released from DYA following either acidic or enzymatic hydrolysis (H89). With a 2-pg sample of DNA, the detection limit for 5-(hydroxymethy1)uracil was found to be 3 molecules lo6 thymine residues. Tandem MS approaches to this prob em also have been explored (H90), and a recent communication from Cooks and co-workers (H91) describes a desorption CI MS/MS method for the determination of OB-methyldeox guanosine from calf thymus DNA which had been exposezin vitro to the methylatin ent methyl methanesulfonate. The recision of the m e t k 3 was reported to be 10% at the 10-R-10-12 mol level, where 0.02% of deoxy anosine residues were methylated. Several papers have Ecribed the use of FAB-MS for the identification (as opposed to the uantitative determination) of nucleic acid adducts. Theselave included reports on the characterization of S-[a-(N-aden 1)ethyllglutathione from digestion of RNA and DNA which l a d been exposed to 1,a-dibromoethane in rat liver cytosol (H92),M-guanine adducts derived from various 4(2-haloethyl)-substitutedcysteine alkylation products and glutathione derivatives (H93, H94), of deoxyguanosine and DNA exposed to the anticancer agent , adducts between phosphoramide mustard thiopeta ( H W and and DNA (Hi%).The technique of hydrogen/deuterium exchange has been found to be useful in facilitating the identification of alkylated bases and other biomolecules (H97)] and MS}MS methods have been shown by FAB-MS (H98), to be capable of distinguishing between isomeric adducts of 7,12-dimethylbenz[a]anthracenein model systems (H99). In a recent mechanistic study of the reaction between deoxyguanosine and both unlabeled and deuterated variants of the anticancer drug BFNU, LC/MS/MS with corona discharge used to identify a series of 7-(2'-haloethyl) anine API It is to be anticipated that t a n e m MS derivatives (H100).
L
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techniques will become widely used in this area of toxicology, and an informative review article on the use of FAB-MS/MS for the identification of carcinogen-nucleoside adducts has been compiled by Lay and co-workers (HIOI). The same oup has reported on the identification by FAB-MS/ MS of &-modified deoxyguanosine and CSmodified deoxyadenosine as major DNA adducts from 2-nitropyrene metabolism in vitro (HI021and has developed general strategies for the analysis of model arylamine-nucleoside adducts using desorption ionization MS, FAB-MS/MS and laser desorption TOF-MS; interestingly, the latter technique was capable of yielding useful structural information from adduct samples as low as 20 fmol (H103). Finally, the study of N-alkylated derivatives of protoporphyrin IX, which are formed by the reaction of prosthetic heme with electrophilic intermediates generated at the active site of cytochrome P-450, has been facilitated by a new method described by De Matteis et al. (H104). Treatment of N-alkylprotoporphyrin IX with Cu2+ions leads to displacement of the alkyl moiety, which may be trapped by a suitable nucleophile, e.g. n-dodecylamine. The resultin alkylated dodecylamine may then be characterized by FA%-MS or FABMS/MS which provides information on the nature of the H106).Reductive meoriginal modified porphyrin (H105, tabolism of the hepatotoxic halocarbon BrCC1, by myoglobin (H107) or hemoglobin (H108) generates free radicals which bind to the heme moiety of the hemoprotein and also lead to cross-linking of the prosthetic oup to the apoprotein. The identities of the adducta formzduring this process have been elucidated by a series of ele ant experiments em loyin FAB-MS, Fourier transform IC&-MSand =2Cf PD-M( whici illustrate effectively the power of mass spectrometry in studies of xenobiotic-derived covalent adducts.
I. STEROIDS, STEROLS,AND BILE ACIDS For many years, mass spectrometry in general, and GC-MS methods in particular, have been used widely in studies of the biosynthesis and metabolism of steroids and bile acids. This continues to be the case, and durin the present reporting period there has been some consolifation of techniques for the qualitative and quantitative analysis of steroids and their derivatives in biological fluids. Thus, GC-E1 MS methods remain the most popular for work with unconjugated steroids (or free steroids liberated from conjugated or fatty acid-esterified metabolites (II)),while FAB-MS and TSP LC-MS are being employed with increasing frequency for the direct analysis of polar steroid con'ugates. Most of these trends are apparent from a chapter by dhackleton, Merdinik, and Lawson (B), which presents an excellent overview of practical approachea to the analysis of steroids and bile acids by MS. The majority of gas-phase techniques for steroid analysis necessitates prior derivatization, and TMS ethers, for which a wide variety of reagents and methods of preparation are available, remain the most commonly used. However, several other of silyl ethers can be prepared readily, and Bertrand et al% have re rted on the results of a comparative study in which the E1 E S properties of cyanoeth ldimethylsilyl (CEDMS), tert-butyldimethylsilyl (t-BDMSY, and TMS ethers of monohydroxy steroids were examined. In was concluded that the structural information content of the mass spectra of the CEDMS ethers (which yield characteristic ions at M - 15, M - 54, and M - 129) was greater than that from the t-BDMS derivatives, and that the most abundant M'+ ions also were obtained with CEDMS derivatives. An interesting application of MS to steroid metabolic profiling is to be found in a publication by Gazdar and coworkers (14) who examined the ability of a human adrenocortical carcinoma cell line grown in vitro to secrete steroids 7-9 years after culture initiation. By means of a combination of GC-MS and LC-MS techniques, it was shown that the cells secreted more than 30 steroids characteristic of adrenocortical cells, including corticosteroids, mineralocorticoids, androgens, and estrogens. Biosynthetic proceases which utilize cholesterol continue to attract attention, and Axelson et al. (15) have demonstrated recently by GC-MS that 7a-hydroxy-3-oxo-4cholestenoic acid is the major metabolite of cholesterol in isolated pig liver mitochondria. Thus,the initial steps in bile acid biosynthesis from cholesterol can take place exclusively in mitochondria, and thereby bypass the rate-limiting cholesterol 7a-hydroxylase reaction which occurs in the endoANALYTICAL CHEMISTRY, VOL. 64, NO. 12, JUNE 15, 1992
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plasmic reticulum. Axelson also has reported that human lasma contains two labile dehydrocholesterolawhich had not with the aid of HPLC-W and k e n detected previously (16); GC-MS methods, one of these sterols was characterized as 5a-cholesta-6,8(9)-dien-3j3-01, while the second was identified tentatively as cholesta-5,8(9)-dien-3~-01.Vitamin D and several of its metabolites have been examined by TSP LC-MS, and a stable isotope dilution TSP asaay has been reported for 1,25-dihydroxyvitamin D3 in human plasma (17). New approaches to the differentiation of isomeric sterols by MS have included collisional activation studies on the M'+ or [M H20]'+ ions formed under E1 conditions. When high-ener (SkeV) collisions were employed and MIKES scans acquirg it was possible to distingulsh 7a- from 78-hydroxycholestero1, a pair of epimeric sterols which afford essentially identical E1 mass spectra (18). Corticosteroids, such as cortisol, traditionally have been considered challenginganalytea due to their low concentrations in biological fluids and also because their thermal instability dictates that GC-MS a proaches include comprehensive derivatization schemes. fn order to obviate these problems, LC-MS methods have been explored for the determination of native corticosteroids in serum. Recently, Paulson and Lindberg (19)reported that acetylation at the (2-21 position, which can be achieved selectively under mild conditions, confers excellent TSP LC-MS properties on cortisol. The limit of detection of cortisol 21-acetate by this approach was estimated to be 0.24 pmol in'ected, which is comparable with many GC-MS methods. drocedures for the detection of synthetic corticosteroids in horse urine have been outlined by Chui et al. (110) who employed LC-MS with atmospheric pressure ionization. Detection limits were on the order of 1 ng mL-', indicating that electrospray or ionspray LC-MS may be especially well-suited to this class of compounds. Despite these developments, GC-MS methods remain valuable for the quantitative determination of low levels of both endo enous and synthetic corticosteroids in biolo 'cal fluids. In t i e case of dexamethasone, a potent and wifely prescribed antiinflammatory corticosteroid, two quite distinct approaches have been described for the measurement of this drug in human plasma. In the first, a tris(TMS) derivative (claimed to be the 17,20-eno1,21 species) is subjected to GC-NI CIMS analysis, when the abundant fragment ion at m / z 446 is monitored; the limit of detection is 0.1 n mL-' from a 1-mL specimen of plasma ( I l l ) . In the seconf method, the dexamethasone is extracted and subjected to oxidation with pyridinium chlorochromate to yield the corresponding 17-keto 1,17-trione steroid, 9a-fluoro-16a-methyl-l,rl-androstadiene-3,1 (112).The latter com ound, which is electrophoric, is then taken for analysis by d)C-NI CIMS, when monitoring the [M - HF]'- anion at m z 310 gives a similar lower limit of detection. Interestin ,a variation on the latter method which emplo ed GC-MjIMS with selected reaction monitoring offereilittle advantage in terms of sensitivity or selectivity, although it was found that the MS/MS approach offered the possibility for sam le introduction via the direct insertion probe, which simpified and shortened the overall analysis (113). As discussed by Teale and Houghton (114),the abuse of anabolic steroids by human athletes and in thoroughbred horse racing is of growing concern to regulatory authorities, and there is an urgent need for im roved methods for the d e m o n and uantitative analysis of &e com unds and their major metaxolites in urine. To this end, Mgbased screening procedures have been developed to detect the administration of anabolic steroids such as nandrolone (114), testosterone (114, nortestosterone (115),1-deh drotestoeterone (114),trenbolone (114,116),and stanozolol $17). In most cases, GC-E1 MS methods were used in these studies, although GC-MS/MS was evaluated by de Boer et al. (116)for the quantification of trenbolone and LC-API MS/MS was employed by Muck and Henion (117) to screen for stanozolol and its urinary metabolites. In support of these dop' control efforts, several investi ations have focused on the Xntification of urinary metabo!ites of commonly-abused anabolic steroids. In almost every case, MS has been employed for structural characterization, and re o r b have appeared on the metabolic fate of methenolone &8), methandienone (119,120),17-methylstenbolone acetate (1211,stanozolol (117, testosterone (119), 122),and boldenone (123).While not an anabolic steroid,
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medroxyprogeaterone acetate also has been of interest in terms of its anticancer activity, and an MS-based study of the metabolic fate of this drug in humans has ap eared (124). Similarly, metabolites of the pharmacologicalyy-active bufadienolide derivative cinobufagin formed in rat liver microsomal re arations have been studied by TSP LC-MS (125). GC-EfM8 was emplo ed by Anderson et al. (126)to study androgen metabolism gy porcine granulosa cells, when it was found that androstenedione is converted by this in vitro system to a 19-carboxylicacid derivative which may serve as a precursor for the formation of C18 neutral steroids. The use of stable isotopes in steroid research has been highlighted in a special two-part issue of the journal Steroids (1274which includes articles describing the use of deuterium labehng and GC-MS in the identification of steroid metabolites in the horse in vitro and in vivo (1281,in the conduct of human pharmacokinetic studies of androgenic steroids and in the (1129)and sulfated rogesterone metabolites (130), development of stabe isotope dilution assay procedures for dehydroe iandrosterone sulfate in human serum by TSP LC-MS (f31)and FAB-MS/MS (132).The contents of this special issue thus provide an effective overview of the scope of stable isotope usage in the steroid field. In the area of bile acid research, MS has been applied to the identification of potential precursors to cholic and chenodeoxycholic acids in man (133)and in cultured human hepatoblastoma cells (134).In the latter paper, a total of 29 cholesterol metabolites were isolated from culture media and cells by liquid-solid extraction and anion-exchange chromatogra hy and were identified by GC-MS as their methyl eater-& derivatives. Similar MS techni ues were employed to identify and quantify abnormal C-1 an! C-6 hydrox latsd bile acids in biolo 'cal fluids from patients with liver &ease (135),while both &-MS and FAB-MS techniques were used by S'ovd and colleagues (136)to identify bile acids and bile alcolols in a child with 38-hydroxy-A5-C,,-steroid dehydrogenase deficiency;direct FAB-MS analysis of specimens of plasma and urine was found to be a simple and effective method to monitor and evaluate the effects of therapy with this infant. In a further clinical study, the formation of recently-described glycosidic conjugates of ursodeo cholic acid was examined by administration of [24-'3C]urs~eoxycholic acid to a healthy volunteer, when both the glucoside and N-acet lglucosaminide con'ugates in urine became enriched in 13C $37). A range of Md techniques, including FAB-MS, TSP LC-MS, and GC-MS, was employed by Nakagawa et al. (138)to determine the biliary acid composition in patients with cystic fibrosis, when more than 50 compounds (mainly glycine and taurine conjugates) were identified. The same grou has reported on the use of GC-MS to study the metaboism of bile acids in the human fetus in early gestation (weeks 13-19) as compared with the full-term fetus whose bile acid profie was determined from the com ition of amniotic fluid (139).Although derivatization of b E c i d s for GC-MS analyais usually entails the formation of methyl esters, Ferreira and Elliott (140)have reported on the preparation and MS characteristics of pentachlorophenyl esters and benzylamide derivatives of the major bile acids, which were analyzed by a number of techniques including TSP LC-MS. A paper has appeared describin the a plication of CF-FAB-MS to metabolic profiling ofbile aci& (1411,and several investigators have examined the utility of tandem MS techni ues to differentiate bile acid isomers; in some cases, TSP L%-MS/MS was used, while FAB-MS/MS was employed in other studies (142-145).Charge-remotefragmentation of taurine-conjugated bile acids has been discussed by Griffiths et al. (1461,who pointed out that such cleavages also have diagnostic value in distinguishing between isomeric bile acids.
J. EICOSANOIDS AND RELATED LIPID MEDIATORS Research on biologically-active lipids remains an extremely active area of study, and MS continues to be the preferred technique for most qualitative and quantitative applications. Due to the constant need for both high sensitivity and high selectivi in the analyak of this claw of compounds, the most common y-used method is electron-ca ture NI CIMS of the ntafluorobenzyl ester derivatives ! t i w sample introduction g"y ca illary GC. Several examples of the application of GCNI C h S to the study of lipid mediators have been discussed
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in a short review by Blair (J1). An interesting development during the present reporting period has been the identification of a series of novel prostaglandin Fz-like compounds in human plasma and urine whose formation is mediated by a non-cyclooxygenase, freeradical-catalyzedmechanism (J2,J3). A variety of techniques was emplo ed to identify these compounds, including GC-NI CIMS, G&EIMS, the use of deuterium-labeled derivatives hydrovenation followed by MS, and immunoreactivity witd an anti-9a,llfl-PGFzantibody. The authors pointed out that formation of these compounds occurs very readily in biological fluids, and therefore it is imperative that methods emplo ed for the quantitative determination of F-series prostaglan$ins be of the highest oasible specificity. Relatively few prostaglandin analogs iave been developed for therapeutic use, although some PGE-like candidates are under investigation tential drugs. Sulprostone, a sulfonamide derivative of is one such compound and a study of its metabolism in human subjects and guinea pigs b GC EIMS and -CIMS has been published (J4).Tandem menods ale0 have been applied to the identification of prostaglandins and thromboxanes. When salts of the carboxylate oup were subjected to FAB-MS and the parent anions cofisionally activated, structurally-informative charge-remote fra entations were Interestingly, observed for many members of this class whereas the carboxylate anion derived from electron capture NI CIMS of the pentafluorobenzyl ester-TMS derivative of PGFk exhibited little fragmentation under low-ener (
