mass spectrometry

Preface Throughout the past decade, mass spectrometry has undergone a dra- matic expansion in experimental methodology and in the structural range of molecules of biological importance to which it can be applied. As a consequence, new approaches and protocols have been added to numer- ous existing mass spectrometry-based methods which have found routine use in the biological sciences. In addition, there are many procedures found in both the mass spectrometry and biochemical literature some of which have undergone subtle but important refinements with time which have never been available in one source. Many new workers wish- ing to assess the capabilities of mass spectrometry or to integrate these methods into the design of biological experiments require a concise and up-to-date treatment of experimental principles and detailed descriptions of specific methods. To address these issues, this volume of Methods in Enzymology is devoted exclusively to mass spectrometry. The first section covers a range of general techniques and topics of contemporary importance in the applications of mass spectrometry to the biological sciences in general. The remaining three sections emphasize specific applications in what is broadly termed structural biology, and are divided among three major classes of biological molecules (peptides and proteins, glycoconjugates, and nucleic acid constituents). Each of these three sections opens with a critical overview of the applications of mass spectrometry to that area, written by a specialist in the field. Coverage of the topics is oriented very strongly, with several excep- tions, toward techniques which have demonstrated value in problem- solving. Some existing newer methods have been excluded because they are still under development and have not yet reached a stage of routine application, although they may ultimately find their way into the reper- toire of routine methods. Also not covered are protocols whose current impact lies primarily in other areas to which mass spectrometry has made major contributions, such as toxicology or environmental science. I am grateful to many of the authors and their colleagues who offered advice on the coverage of specific topics, including the interrelatedness of certain chapters. Particular appreciation is expressed to Klaus Biemann, Roger A. Laine, and Robert C. Murphy for their critical advice on the organization of the volume and on the contents of specific chapters. JAMES A. MCCLOSKEY xiii Contributors to Volume 193 Article numbers are in parentheses following the names of contributors. Affiliations listed are current. ANNE-SoPHIE ANGEL (32), BioCarb Tech- nology AB, S-22370 Lund, Sweden KALYAN R. ANUMULA (27), Department of Analytical Chemistry, SmithKline Bee- cham Pharmaceuticals, King of Prussia, Pennsylvania 19406 JOHN R. BARR (27), Department of Physical and Structural Chemistry, SmithKline Beecham Pharmaceuticals, King of Prus- sia, Pennsylvania 19406 KLAUS BIEMANN (13, 18, 25, A.5, A.6), De- partment of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139 ROaERT K. BOYD (7), Institute for Marine Biosciences, National Research Council, Halifax, Nova Scotia B3H 3Z1, Canada A. L. BURLINGAME (36, 37), Department of Pharmaceutical Chemistry, School of Pharmacy, University of California-San Francisco, San Francisco, California 94143 RICHARD M. CAPRIOLI (9), Analytical Chemistry Center, and the Department of Biochemistry and Molecular Biology, University of Texas Medical School at Houston, Houston, Texas 77030 STEVEN A. CARR (27), Department of Physi- cal and Structural Chemistry, SmithKline Beecham Pharmaceuticals, King of Prus- sia, Pennsylvania 19406 KEITH L. CLAY (17), Department of Pediat- rics, National Jewish Center for Immu- nology and Respiratory Medicine, Den- ver, Colorado 80206 PHILIP COHEN (26), Department of Bio- chemistry, University of Dundee, Dundee DD1 4HN, Scotland CATHERINE E. COSTELLO (40, A.3), Mass Spectrometry Facility, Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139 ROBERT J. COTTER (1), Department of Phar- macology, Middle Atlantic Mass Spec- trometry Facility, The Johns Hopkins University School of Medicine, Balti- more, Maryland 21205 PAMELA F. CRAIN (42, 47), Department of Medicinal Chemistry, University of Utah, Salt Lake City, Utah 84112 ANNE DELL (35), Department of Biochemis- try, Imperial College of Science, Technol- ogy, and Medicine, London SW7 2AZ, England EDWIN OE PAUW (8), Department of Chem- istry, Liege University, B-4000 Liege, Belgium MIRAL DIZDAROGLU (46), Center for Chem- ical Technology, National Institute of Standards and Technology, Gaithers- burg, Maryland 20899 GREGORY G. DOLNIKOWSKI (2), USDA Hu- man Nutrition Research Center at Tufts University, Boston, Massachusetts 02111 BRUNO DOMON (33), Physics Department, Ciba-Geigy Ltd., CH-4002 Basel, Switzer- land CHARLES G. EDMONDS (22), Chemical Sci- ences Department, Pacific Northwest Laboratory, Richland, Washington 99352 HEINZ EGGE (38), lnstitut far Physiolog- ische Chemie, Universitdt Bonn, D-5300 Bonn 1, Federal Republic of Germany JAMES I. EELIOTT (21), Department of Mo- lecular Biophysics and Biochemistry, Yale University, New Haven, Connecti- cut 06510 SYO EVANS (3), Kratos Analytical Instru- ments, Urmston, Manchester M31 2LD, England ix X CONTRIBUTORS TO VOLUME 193 BRADFORD W. GIBSON (26), Department of Pharmaceutical Chemistry, School of Pharmacy, University of Caiifornia at San Francisco, San Francisco, Cal- ifornia 94143 BETH L. GILLECE-CASTRO (37), Department of Pharmaceutical Chemistry, School of Pharmacy, University of California at San Francisco, San Francisco, California 94143 GARY R. GRAY (31), Department of Chemis- try, University of Minnesota, Minneapo- lis, Minnesota 55455 MICHAEL L. GROSS (6, 10), Midwest Center for Mass Spectrometry, Department of Chemistry, University of Nebraska, Lin- coln, Nebraska 68588 GUNNAR C. HANssON (39), Department of Medical Biochemistry, G6teborg Univer- sity, S-400 33 G6teborg, Sweden ALEX G. HARRISON (1), Department of Chemistry, University of Toronto, To- ronto, Ontario M5S IAI, Canada ROGER N. HAYES (10), Department of Chemistry, Midwest Center for Mass Spectrometry, University of Nebraska, Lincoln, Nebraska 68588 CARL G. HELLERQVIST (30), Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232 FRANZ HILLENKAMP (12), lnstitutfar Medi- zinische Physik, Universitiit Manster, D-4400 Miinster, Federal Republic of Germany IAN JARDINE (24), Analytical Biochemistry Division, Finnigan MAT, San Jose, Cali- fornia 95134 KEITH R. JENNINGS (2), Department of Chemistry, University of Warwick, Cov- entry CV4 7AL, England MICHAEL KARAS (12), Institut fiir Medizin- ische Physik, Universitiit Miinster, D-4400 M~inster, Federal Republic of Germany HASSE KARLSSON (39), Department of Med- ical Biochemistry, GOteborg University, S-400 33 GOteborg, Sweden KARL-ANDERS KARLSSON (34), Department of Medical Biochemistry, University of G6teborg, S-400 33 GOteborg, Sweden DANIEL R. KNAPP (15), Department of Cell and Molecular Pharmacology and Experi- mental Therapeutics, Medical University of South Carolina, Charleston, South Carolina 29425 ROGER A. LAINE (29), Departments of Bio- chemistry and Chemistry, Louisiana State University and The LSU Agricul- tural Center, Baton Rouge, Louisiana 70803 TERRY D. LEE (19), Division of Immunol- ogy, Beckman Research Institute of the City of Hope, Duarte, California 91010 RONALD D. MACFARLANE (11), Department of Chemistry, Texas A&M University, College Station, Texas 77843 STEPHEN A. MARTIN (28), Genetics Insti- tute, Andover, Massachusetts 01810 JAMES A. McCLOSKEY (16, 41, 44, 45, A.1, A.4), Department of Medicinal Chemis- try, University of Utah, Salt Lake City, Utah 84112 WALTER McMoRRAY (21), Comprehensive Cancer Center, Yale University, New Ha- ven, Connecticut 06510 RICHARD M. MILBERG (14, A.2), School of Chemical Sciences, University of Illinois, Urbana, Illinois 61801 WtLLtAM T. MOORE (9), Analytical Chemistry Center, and the Department of Biochemistry and Molecular Biology, University of Texas Medical School at Houston, Houston, Texas 77030 DIETER R. MOLLER (33), Physics Depart- ment, Ciba-Geigy Ltd., CH-4002 Basel, Switzerland ROBERT C. MURPHY (17), Department of Pediatrics, National Jewish Center for Immunology and Respiratory Medicine, Denver, Colorado 80206 BO NILSSON (32), BioCarb Technology AB, S-22370 Lund, Sweden JASNA PETER-KATALINIC (38), Institut fiir Physiologische Chemie, Universitiit Bonn, D-5300 Bonn 1, Federal Republic of Germany CONTRIBUTORS TO VOLUME 193 xi GLENN PETERSON (21), Adirondack Envi- ronmental Services, Albany, New York 12207 WESTON PIMLOTT (34), Department of Med- ical Biochemistry, University of G6te- borg, S-400 33 G6teborg, Sweden STEVEN C. POMERANTZ (44), Department of Medicinal Chemistry, University of Utah, Salt Lake City, Utah 84112 LINDA POULTER (36), Biotechnology De- partment, ICI Pharmaceuticals, Cheshire SKIO 4TG, England WILHELM J. RICHTER (33), Physics Depart- ment, Ciba-Geigy Ltd., CH-4002 Basel, Switzerland GERALD D. ROBERTS (27), Department of Physical and Structural Chemistry, SmithKline Beecham Pharmaceuticals, King of Prussia, Pennsylvania 19406 PETER ROEPSTORFF (23), Department of Molecular Biology, Odense University, DK 5230 Odense M, Denmark Bo E. SAMUELSSON (34), Department of Medical Biochemistry, University of GOteborg, S-400 33 G6teborg, Sweden KARL H. SCHRAM (43), Department of Pharmaceutical Sciences, College of Pharmacy, University of Arizona, Tuc- son, Arizona 85721 HUBERT A. SCOBLE (28), Genetics Institute, Andover, Massachusetts 01810 JOHN E. SHIVELY (19), Division of Immu- nology, Beckman Research Institute of the City of Hope, Duarte, California 91010 DAVID L. SMITH (20), Department of Me- dicinal Chemistry and Pharmacognosy, Purdue University, West Lafayette, Indi- ana 47907 RICHARD D. SMITH (22), Chemical Sciences Department, Battelle, Pacific Northwest Laboratory, Richland, Washington 99352 KATHRYN L. STONE (21), Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecti- cut 06510 PAUL B. TAYLOR (27), Department of Mac- romolecular Sciences, SmithKline Bee- cham Pharmaceuticals, King of Prussia, Pennsylvania 19406 JAMES E. VATH (40), Genetics Institute, An- dover, Massachusetts 01810 MARVIN L. VESTAL (5), Vestec Corpora- tion, Houston, Texas 77054 J. THROCK WATSON (4), Departments of Biochemistry and of Chemistry, Michigan State University, East Lansing, Michigan 48824 KENNETH R. WILLIAMS (21), Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecti- cut 06510 RICHARD A. YOST (7), Department of Chemistry, University of Florida, Gaines- ville, Florida 32611 ZHONGRUI ZHOU (20), Department of Me- dicinal Chemistry and Pharmacognosy, Purdue University, West Lafayette, Indi- ana 47907 [1] METHODS OF IONIZATION 3 [1] Methods of Ionization By ALEX G. HARRISON and ROBERT J. COTTER Introduction Historically, in the development and application of mass spectrometry to the analysis of organic molecules, during the 1950s and early 1960s, electron ionization (EI) was the only practical ionization method available. The development of chemical ionization (CI) in the late 1960s provided a complementary method for ionizing gaseous molecules. A disadvantage of both methods is that the sample of interest must be present in the gas phase at a pressure of 10 -5 to 10 -4 tOIT. With the extension of mass spectrometry to large, involatile, and, often, thermally fragile biomolec- ules, research during the 1970s and 1980s has been directed toward the development of ionization methods capable of ionizing such molecules directly from the solid or solution state; this work has led to the develop- ment of a variety of de sorption ionization techniques. This chapter reviews these various methods of ionizing molecules in the gas phase and directly from the liquid or solid state. Electron Ionization Basic Principles When gaseous polyatomic molecules are ionized by interaction with a beam of electrons the production of ions can be classified, phenomenologi- cally, under the following schemes: Ionization ABC + e-*ABC ÷ + 2e Dissociative Ionization Ion-Pair Formation Electron Capture ABC+ e *AB + + C+2e ~ AC + + B + 2e ABC + e >AB + + C- + e ABC +e~ABC- METHODS IN ENZYMOLOGY, VOL, 193 (1) (2a) (2b) (3) (4) Copyright © 1990 by Academic Press, Inc. All fights of reproduction in any form reserved. 4 GENERAL TECHNIQUES [1] 1600 - 1400 - 1200 r'r n,- Z t ,_, 0 Z ~ uJ D I000400600200800 -/~ 0 I0 20 CH30H + co. I I 30 40 50 60 70 80 90 I00 ELECTRON ENERGY (eV) FIG. 1. Ionization efficiency curves for major ions in the mass spectrum of methanol. (From Ref. 4, with permission.) Dissociative Electron Capture ABC + e~AB- + C (5) Under normal operating conditions (20-70 eV electron energy) reactions (I) and (2a,b) account for the major part of the ionization with, in many cases, a minor contribution from reaction (3). In the electron-capture reactions (4) and (5) there are no product electrons to carry away the excess energy; hence, these reactions are resonance processes which have significant cross sections only over a very narrow range of electron energies, usually in the range 0-10 eV, i.e., normally below the threshold for processes (1) and (2). As a result, the literature of electron ionization is primarily concerned with positive ion mass spectra; for a discussion of electron-capture negative ion mass spectrometry, see later in this chapter. Reaction (1) occurs when the energy of the bombarding electrons exceeds the ionization energy of the molecule. Typically a greater electron energy is required for dissociative ionization to take place because of the energy required to break chemical bonds either in simple bond rupture [reaction (2a)] or in bond rupture accompanied by rearrangement [reaction (2b)]. Typical ionization efficiency curves are shown in Fig. 1 for the major [1] METHODS OF IONIZATION 5 B FIG. 2. Electron ionization source. F, Filament; B, source block; T, trap; R, repeller. ions in the mass spectrum of the simple molecule CH3OH. Of particular interest is the broad plateau in the ion yields between about 50 and 100 eV ionizing electron energy. In this region both the total ion yield and the relative ion abundances are relatively insensitive to the ionizing electron energy employed. Consequently, reproducible mass spectra and a con- stant sensitivity can be achieved by operating in this plateau region. By contrast, operating below 20-30 eV electron energy results in mass spectra and sensitivities that are strongly dependent on the ionizing electron energy. In the plateau region, the total positive ion current, I+, produced in the ion source is given by I+ = Qil[N]Ie (6) where Qi is the total ionization cross section, I is the ionizing path length, [N] is the concentration of neutral molecules per cm 3, and Ie is the ionizing electron current. Measured ionization cross sections lie in the range 0.42 × 10-16 cm 2 for helium to 30 x 10- ~6 cm 2 for n-decane~; for more complex molecules higher cross sections can be expected since molecular cross sections are approximately an additive function of atomic ionization cross sections 2 or, alternatively, increase roughly linearly with increasing molec- ular polarizabilities) Instrumentation A schematic diagram of a typical electron ionization source is shown in Fig. 2. Electrons, emitted from an electrically heated filament, are accelerated through the necessary potential (usually 70 V) and introduced into the ionization region where a small fraction interact with the gaseous sample molecules; the remainder impinge on the trap electrode. Usually a constant ionizing electron current is maintained by a feedback circuit to the filament power supply to maintain a constant trap current. A positive l A. G. Harrison, E. G. Jones, S. K. Gupta, and G. P. Nagy, Can. J. Chem. 44, 1967 (1966). 2 j. W. Otvos and D. P. Stevenson, J. Am. Chem. Soc. 78, 549 (1956). 3 F. W. Lampe, J. L. Franklin, and F. H. Field, J. Am. Chem. Soc. 79, 6129 (1957). 6 GENERAL TECHNIQUES [1] voltage, with respect to the cage, applied to the repeller electrode helps remove the ions for mass analysis; there often are other electrodes (not shown) to extract, shape, and guide the ion beam before entry into the mass analyzer. This type of ion source can be employed with any of the mass analyzers discussed in [2] of this volume, although with the time-of- flight (TOF) analyzer it is necessary to pulse the ionizing electron beam and/or the extraction lenses. The sample to be ionized must be in the gas phase at a pressure in the ion source of less than 10 -4 torr; higher pressures will lead to ion/molecule reactions between the primary ions and the neutral molecules. Such reac- tions can distort the mass spectrum by producing new species such as the protonated molecule, MH ÷ . This problem is more severe for combined EI/CI sources which, even when operated in the E1 mode, usually are more gas-tight than sources designed for EI operation only, thus producing a higher ion source pressure for the same pressure reading on a remote pressure gauge. The sample may be introduced as a gas from a heated inlet system, by evaporation from a direct insertion probe, or as the effluent from a gas chromatograph. Using Eq. (6) it can be derived that a pressure of 10 -5 torr of a sample with Qi 100 × 10 -16 cm 2 will produce a total source ion current of approximately 10 -7 A for a typical ionizing electron current of 100/.~A and a source temperature of 150 °. Since this ion current is spread over many m/z values and it may be desired to detect a minor component in a gas mixture, the need for sensitive ion detection is evident. This is particularly true for sector instruments where 1% or less of the ions formed in the ion source reach the detector. In this respect, quadrupole analyzers have the advantage of a much higher percentage ion transmission. Origin of Electron Ionization Mass Spectra To reach some understanding of the factors which determine the final EI mass spectrum observed it is necessary to consider briefly and qualita- tively the mechanisms by which molecular and fragment ions are formed. A more comprehensive discussion can be found elsewhere. 4-6 For polyatomic molecules it is well-established that the ionization step is separate in time from the decomposition reactions which give rise to the 4 A. G. Harrison and C. W. Tsang, in "Biochemical Applications of Mass Spectrometry" (G. R. Waller, ed.), pp. 135-156. Wiley, New York, 1972. 5 M. E. Rose and R. A. W. Johnstone, "Mass Spectrometry for Chemists and Biochemists." Cambridge Univ. Press, New York, 1982. 6 I. Howe, D. H. Williams, and R. D. Bowen, "Mass Spectrometry, Principles and Applica- tions." McGraw-Hill, New York, 1981. [1l METHODS OF IONIZATION 7 fragment ions. The electron/neutral interaction occurs in the order of 10-16 sec and leads to formation of molecular ions with various amounts of excitation energy (electronic and vibrational) with respect to the ground state of the molecular ion. The range of excitation energies is determined by the accessible states and the transition probabilities to these states and leads to a distribution of internal energies of the molecular ions which may extend to 10-20 eV. The electronic states formed are nonrepulsive and have a significant lifetime during which the excess internal energy is ran- domly redistributed among the vibrational degrees of freedom of the ground electronic state. Since ion/neutral collisions are negligible these energy randomization processes occur intramolecularly, primarily by radi- ationless transitions at the numerous crossings of potential energy sur- faces. Decomposition of a molecular ion occurs whenever sufficient energy accumulates in the appropriate vibrational mode or modes to cause bond rupture. The fragment ions formed may have sufficient internal energy to fragment further and rearrangement of the molecular framework may occur at any time. The reactions leading to the formation of a mass spec- trum, thus, are a series of competing and consecutive unimolecular decom- position reactions originating from the molecular ion, the rate of each reaction being dependent on the internal energy. In principle, the rate constant for each step can be calculated by applying an appropriate form of the absolute reaction rate theory. In their exact formulation and quantitative application to the origin of E1 mass spectra the theory of rate processes, whether the quasi-equilib- rium theory (QET) 7 or the similar Rice, Ramsperger, Kassel, and Marcus (RRKM) theory, 8 is highly physical and mathematical in approach. A less precise approach which leads to at least a qualitative understanding of the factors which determine the appearance of mass spectra leads 4-6 to Eq. (7) for the rate constant for an ionic fragmentation reaction k(E) = v[(E - Eo)/E] s (7) where v is a frequency factor, E the internal energy of the ion, E 0 the activation energy (or critical reaction energy), and S is the effective num- ber of oscillators [often taken as one-half to one-third the total number of oscillators (3N-6 for an N-atom molecule)]. The frequency factor v is effectively a measure of the entropy of activation. For a simple bond rupture reaction it usually is taken as the frequency of a bond vibration; 7 H. M. Rosenstock, M. B. Wallenstein, A. L. Wahrhaftig, and H. Eyring, Proc. Natl. Acad. Sci. U.S.A. 38, 667 (1952). s R. A. Marcus, J. Chem. Phys. 20, 359 (1952). 8 GENERAL TECHNIQUES [1] 12 v _J 4 12 _I (b) (c) II Eo:2 , ,~ :10 j4 Eo:2 , 0:10 a° 4 o o l I I I 1 J I I I 2 4 6 8 INTERNAL "-' Eo - Eo: l, ,~:10 IO I I I I I0 12 14 16 ENERGY (eV) FIG. 3. Internal energy distribution and log k(E) versus E curves, v = 1014 is characteristic of a simple cleavage, while v = 101° is characteristic of a rearrangement. however, for a rearrangement reaction involving a particular spatial ar- rangement of atoms the entropy of activation will be less favorable and this is reflected in a lower frequency factor. Equation (7) leads to rate constants which increase rapidly with increas- ing internal energy to a limiting value determined by the value of v. Two curves are shown in Fig. 3b where E0 is the same but different frequency factors are invoked corresponding qualitatively to a simple bond rupture (v = 1014 sec -l) and a rearrangement reaction (v = 10 l° sec-l). Figure 3a shows a hypothetical, but plausible, distribution of internal energies deposited in the molecular ions in the initial ionization step. A consequence of the rate formulation is that an assembly of molecular [...]... fragmentation High -Mass Range PDMS has been the most effective, routine method for obtaining molecular masses of peptides in the 5-20 kDa range Used in conjunction with TOF mass analyzers, the resulting low-resolution mass spectra can nevertheless provide accurate mass measurements,57 since they are generally uncomplicated by fragmentation Molecular mass measurements by FAB mass spectrometry have been... thiolglyclrol, DEAl FiG 6 An overview of particle beam techniques (A) Plasma desorption mass spectrometry (PDMS); (B) secondary ion mass spectrometry (SIMS); (C) fast atom bombardment (FAB) a surface and release sample, or secondary ions However, the term secondary ion mass spectrometry (SIMS) has been used primarily to designate mass spectrometric methods in which the primary ion beam has energies in the kiloelectron... energy for further fragmentation of a primary fragment ion is low, the primary fragment ion will be of low abundance in the mass spectrum even though the reaction to form this ion may be favorable Finally, it should be noted that it is common in organic mass spectrometry to rationalize mass spectra in terms of charge localization and the rupture of adjacent bonds to give stable fragmentation products, 10,11particularly... methods were used for the sputtering of metal ions from surfaces, and not for the desorption of intact molecular ion species Plasma Desorption Mass Spectrometry Ionization of heavy molecules using multiply charged megaelectron volt ions is known as plasma desorption mass spectrometry (PDMS) 5n In this technique, energetic particles are generated from the spontaneous fission events occurring in a 10/zCi sample... foil on which the sample is deposited (Fig 6) Sample ions are extracted by an electrical field and mass analyzed by a TOF mass spectrometer In addition to its high mass range and high ion transmission, the TOF analyzer is the most practical, since the low ionization rates essential to this method preclude mass- scanning techniques Particles in the same 1 MeV/dalton range have also been generated 51 R D... resulting mass spectra are generally dominated by atomic ions, nonspecific fragment ions, and [in the case of fast atom bombardment (FAB)] matrix ions in the low mass region, desorption spectra provide structurally informative molecular and fragment ions in the high mass region Mechanisms A interesting feature of desorption methods is that a wide array of instrumentally different approaches result in mass. .. Similarly, [M + C3H 3] + and [M + C4H9] + ions sometimes may be observed with low intensities in isobutane CI mass spectra It also should be noted that, in H 2 and C H 4 CI mass spectra, [M - H] ÷ ions may be observed as the result of hydride abstraction; these are particularly prominent in H E CI mass spectra and, in the absence of abundant MH ÷ ion signals, may be mistakenly identified The question may... of the fragment ion signals to the high mass side It will be noted that the abundance of the molecular ion in the mass spectrum is determined largely by the activation energy, E0, for the lowest energy fragmentation reaction and by the fraction of molecular ions with internal energies below E0 Two situations can lead to the absence of the molecular ion in the mass spectrum: (a) the activation energy... use in surveying the carbohydrate heterogeneity in the high mannose glycopeptide from the variant surface glycoprotein (VSG) from trypanosomes.58 The mass resolution is low; however, the measured molecular ion masses correspond well to the calculated masses for a glycopeptide: Phe-Asn(GlcNAc2Mann)-GluThr-Lys, containing from 5 to 9 mannose units M o l e c u l a r S e c o n d a r y I o n M a s s S p... monolayer on a silver surface so that the predominant molecular ion species are [M + Ag] ÷ ions While early experiments using quadrupole mass analyzers limited the mass range of this technique, both sector instruments 6° and TOF analyzers 6L62 have been utilized for high mass analysis F a s t A t o m B o m b a r d m e n t Fast atom bombardment (FAB) 46 employs a neutral 10 keV Ar (or Xe) beam to sputter . numer- ous existing mass spectrometry- based methods which have found routine use in the biological sciences. In addition, there are many procedures found in both the mass spectrometry and biochemical. devoted exclusively to mass spectrometry. The first section covers a range of general techniques and topics of contemporary importance in the applications of mass spectrometry to the biological. Scotland CATHERINE E. COSTELLO (40, A.3), Mass Spectrometry Facility, Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139 ROBERT J. COTTER (1),