ASMS Sizzles in Tucson
W
ith the mercury topping 115 °F in Arizona's Sonoran Desert, 1820 mass spectrometrists gathered for the 38th American Society for Mass Spectrometry (ASMS) Conference on Mass Spectrometry and Allied Topics, held June 3-8. Because of its size, this year's scientific program convened at two sites in the Old Pueblo. Each morning session began at the Westin La Paloma Resort with a plenary lecture followed by four concurrent sessions made up of symposia and oral presentations. After lunch, conferees shuttled to Loew's Ventana Canyon Resort for an afternoon of poster sessions and workshops in which new information could be exchanged in a relaxed atmosphere. Organizers of the program succeeded in a difficult task: accommodating the varied interests of the participants. ASMS, formed in 1969, promotes and disseminates knowledge about MS and allied topics. The society's approximately 3000 U.S. and foreign members are involved in research and development, and their interests include the advancement of techniques and instrumentation in MS as well as fundamental research in chemistry, geology, biological science, and physics. Sessions at the conference spanned such diverse topics as LC/MS, isotopic techniques, FT-MS and ion traps, elemental MS, ion chemistry and ion physics, tandem MS and activation methods, hybrid MS, and state-selected chemistry. Clearly evident in the program and the meeting discussions was the MS community's excitement about charac-
terizing large biomolecules. Advances in biotechnology have necessitated extremely sensitive, precise, and accurate methods of analysis. Until recently these analyses were obtained primarily by chromatographic and electrophoretic techniques; MS methods suffered from difficulties in ionizing large, nonvolatile, and labile molecules. Some of these obstacles have been overcome by recent advances in electrospray (ES) ionization and matrix-assisted laser desorption (LD). Monday's plenary lecture by Franz Hillenkamp of the Universitât Munster (FRG) described work done at his laboratory to desorb proteins of masses as high as several hundred thousand
FOCUS daltons by UV and IR laser desorption/ ionization. The analyte is highly diluted in a matrix (typically, nicotinic acid) that absorbs strongly at the laser wavelength. This solution is placed on a metallic substrate and air dried. Ions are then generated by a Q-switched frequency quadrupled Nd-YAG laser and analyzed with a time-of-flight (TOF) mass spectrometer. The resulting spectra consist of peaks from ions of parent species (usually singly charged) and have little or no fragmentation. Hillenkamp also described experiments using an IR laser and showed the spectrum of monoclonal antibody IgG desorbed with a TEA-C0 2 laser. Hillenkamp's group has obtained
spectra of glycoproteins with a carbohydrate content > 20%, as well as spectra of polynucleotides and underivatized oligosaccharides with molecular masses of 40 000 Da and 5000 Da, respectively. Low picomole-to-femtomole amounts of sample are needed for preparation, and the amount of sample consumed is estimated to be in the attomole range. Because the technique appears to be independent of molecular properties such as polarity and surface activity, it is also useful for mixture analysis. The accuracy of the mass determination is estimated to be in the 10-3-10-4 range. Following Hillenkamp's plenary lecture, Brian Chait of The Rockefeller University chaired an extremely wellattended symposium on the generation of high-mass ions by ES ionization and by LD. ES ionization of high-mass biomolecules produces a distribution of highly charged molecular ions that, depending on the molecule, can contain tens to hundreds of charges. In the first talk of the session, John Fenn of Yale University addressed the question of how large a molecule can be transformed intact into ions via ES. He noted that reports of ES ionization for biopolymers presented at the 1988 ASMS meeting described mass analyses of proteins with molecular weights as high as 40 000. Within a few months, ions with masses > ~ 133 000 Da had been obtained, and results with much larger masses have since been realized. He cautioned, however, that until ionization mechanisms are better understood, values for molecular weight lim-
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FOCUS its and charge multiplicity cannot be predicted and must rely on experimental results. Fenn reported results suggesting that ES can produce ions with molecular weights up to 5 million, although the usefulness of this capability remains to be seen. Because of charge multiplicity in such large ions, the spectral peaks cannot yet be resolved by available analyzers. To date, much of the ES work has been carried out with quadrupole mass filters and triple quadrupole mass spectrometers. However, other mass analyzers can be used, such as those described by Gary J. Van Berkel of Oak Ridge National Laboratory. His group recently coupled an ES source with an ion trap mass spectrometer, which has a nominal m/z range of 650. By using a technique called axial modulation, they were able to extend the mass range by as much as 10-fold. They reported that they had obtained spectra of myoglobin (16 950 Da), bovine albumin (66 000 Da), yeast alcohol dehydrogenase (150 000 Da), and bovine thyroglobulin (669 000 Da). Many mass spectrometrists are eager to incorporate ES sources into their laboratories but are unable to justify the cost of a complete new ESMS system. To address this problem, Ian Jardine of Finnigan MAT described a source available from Analytica of Branford that can be easily retrofitted into differentially pumped quadrupole mass spectrometers. This source has been used successfully with picomole sensitivity to obtain protein molecular weights with high accuracy; to sequence peptides by MS/MS of singly, doubly, or triply protonated precursor ions; and to detect and identify peptides and proteins eluting from separation systems such as capillary electrophoresis and LC. Retrofit ES sources are also available from Vestec Corp. ES sources can also be used with other mass analyzers. Barbara Larsen of Du Pont and Matthias Mann of Odense University (Denmark), among others, presented results obtained by mass analysis of ES ions with magnetic sector instruments. Kent Henry of Cornell University described the combination of an ES source with an FT-ICR apparatus. Several of the presenters concentrated on applications made possible by ES. For example, Jack Henion of Cornell University pointed out that until recently, characterizing glycoproteins and glycopeptides has been a difficult analytical challenge. However, by coupling an LC separation on line with pneumatically assisted ES (ion spray) LC/MS and a triple quadrupole mass spectrometer, one can collect molecu-
lar weight and structural information about each component in the chromatogram. Molecules that have been studied by Henion's group include tissue plasminogen activator, renin, and several oligosaccharides. Rong Feng of the National Research Council of Canada described his group's work in determining the mass of horse myoglobin (16 950.4 Da) to within ± 0.2 Da (precision 12 ppm). At this level of accuracy, his group determined that two iV-terminal variants (MW 7416.2 ± 0.3 and 7531.3 ± 0.1) of the plant toxin cerato ulmin were missing Ser and Ser-Asp, respectively, from the precursor ion (MW 7618.4 ± 0.3). The MW of bovine serum albumin (BSA) was determined to be 66 432 ± 2.6 (precision 39 ppm), rather than the calculated value of 66 264, suggesting the possibility of post-translational modification in BSA. Feng also described an ESMS method for precise determination of the number of disulfide bridges and free thiol groups in proteins. For example, j8-lactoglobulin was shown to have 2.0 S-S bonds and 1.0 free -SH group,
44 Each technique has specific strengths and limitations. %% whereas lysozyme has 4.0 S-S bonds and no free -SH group. Feng's group has also used ESMS to differentiate covalent and noncovalent bindings in enzyme-inhibitor interactions and to determine the number of covalent binding sites and their relative affinities to the inhibitor. Joseph Loo of Battelle's Pacific Northwest Laboratory discussed his group's work with ES ionization in combination with tandem MS and collisionally activated dissociation (CAD) to yield structural information about large intact proteins. Dissociation of each multiply charged molecular ion yields fragment ions that can be related to the primary structure (amino acid sequence) of the polypeptide. In most of the cases studied (molecules ranging in size from peptides to large proteins up to 66 kDa), multiply charged product ions result from bond breaking near the ends of the parent ion and are detected with a triple quadr u p l e instrument. More than 30 other papers on ES ionization were presented, reflecting
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the start-up ease and also the value of this new technique. Lasers also play an important role in characterizing large biomolecules, and several talks focused on their use in generating high-mass ions. Peter Williams of Arizona State University described a general volatilization technique whereby large biomolecules are transported into the vapor phase by an expanding vapor plume generated by pulsed-laser ablation of frozen aqueous solutions of analytes. Using a TOF mass spectrometer, his group has determined that positive ions of nucleic acid analytes are generated upon ablation, that the ionization efficiency depends on laser power density and wavelength, and that fragmentation of the parent molecule varies inversely with the laser power density. By using an electron multiplier without postacceleration, Williams and Randall Nelson have observed molecular ions of the DNA transcription terminators UTT (16 base pairs, 10 500 Da) and trpA (28 base pairs, 18 500 Da). Autoradiographic detection of ablated intact DNA up to 6 MDa suggests that this approach to characterizing DNA is limited only by the mass range of available detectors. The introduction of matrix-assisted UV-LD by Karas and Hillenkamp in 1988 has prompted other researchers to investigate possible applications of the technique and the mechanisms involved. Werner Ens of the University of Manitoba noted that the spectral quality improves as the laser power density decreases until a threshold for observation is reached. He described work to reduce the observation threshold and to explore the desorption process itself. Results from the Manitoba group's work demonstrate that a true production threshold exists (~ 106 W/ cm2) and that desorption is a collective effect involving at least 103 analyte ions per pulse with the normal analyte concentration in the matrix (1:1000). By reducing the analyte concentration, useful spectra were obtained with a few tens of ions desorbed per pulse. Considerable interest at the conference also focused on the kinetic energies of ions produced by this method, because this affects resolution and detection in TOF analyzers. Bernhard Spengler of the University of Dusseldorf (FRG) reported measurements of kinetic energy distributions of ionized protein in the mass range 15 000 to 70 000 Da as high as 20% of the nominal acceleration voltage. He also showed that this range could be narrowed by two-stage acceleration. Y. Pan of Johns Hopkins University described analyses of proteins above
The American Chemical Society presents a n i n t e n s i v e 4 - d a y Short C o u r s e 116 000 Da on a Wiley-McLaren TOF instrument. She reported that the delayed draw-out pulse on this type of mass spectrometer could be used to measure mass-dependent kinetic energy spreads and to select ions for analysis with narrower energy distributions. Monday's symposium also covered applications of matrix-assisted UVLDMS. Ronald Beavis of The Rockefeller University detailed the use of this technique for direct examination of complicated mixtures of proteins found in unpurified biological fluids such as blood plasma, cell lysates, and mammalian milk. By using 3,5-dimethoxy-4-hydroxycinnamic acid as a matrix material and a laser wavelength of 354.6 nm, the Rockefeller group obtained molecular mass information about each protein component of a mixture with a mass resolution of 300500 and a mass accuracy of 0.01%. Contaminants such as buffers and salts at high concentrations (0.1 M) that normally affect MS analyses of proteins, as well as nonionic contaminants such as lipids and oligosaccharides, do not appear to interfere with the analysis. According to these researchers, the measurement of such complex mixtures is unprecedented in MS. Given the merits of the ES and matrix-assisted LD approaches for characterizing proteins, how does one choose between the two for a particular application? In the final talk of Monday's symposium, Brian Chait noted that this type of comparison has not been undertaken previously because of the relative novelty of both approaches. For the past six months, his research group has operated both a laser desorption T O F mass spectrometer and an ES source for a quadrupole mass analyzer. This experimental arrangement allowed them to compare results obtained by both techniques on the same samples. Based on this information, Chait concludes for the time being that the methods appear to be complementary—each technique has specific strengths and limitations. Both appear to have similar mass accuracies (0.01%) and produce masses that agree within that precision on standard protein samples. They find ES to be preferable for mixtures of tryptic peptides and in cases where CAD MS/MS techniques are important. On the other hand, LDMS is preferable when the amount of sample is limited or when the samples under study contain contaminants, mixtures of proteins, or unknown proteins. The emphasis on large molecules was echoed on Wednesday morning when ASMS president Ronald Hites of Indiana University announced the estab-
lishment of the ASMS Award for Distinguished Contribution to Mass Spectrometry and presented it to Ronald Macfarlane of Texas A&M University. The annual award recognizes focused achievement in or contribution to fundamental or applied MS and is intended to acknowledge a single, unique contribution to the field. Recipients receive a plaque and a cash award of $2000. Macfarlane was honored for his work on the conception, development, and application of 252Cf plasma desorption MS. While studying beta-emitting nuclei at the cyclotron laboratory at Texas A&M in 1973, Macfarlane noticed that the ions detected in the process did not correlate with beta decay. He concluded that atoms and molecules on a foil surface (which were probably hydrocarbons and salt contaminants) were being ionized and emitted in the process. Recognizing the potential of this observation, Macfarlane used fission fragments from 252Cf rather than beta emitters to deposit more energy on the surface of a sodium acetate target and used a TOF analyzer for mass analysis. This experiment produced the first 252Cf plasma desorption mass spectrum showing high-intensity atomic hydrogen, sodium, and aluminum ions as well as fragments of the acetate moiety. In the years following this discovery, Macfarlane and others have developed instrumentation and sample preparation techniques for the analysis of underivatized organic molecules. They have successfully expanded the mass range for desorbed molecules to more than 30 000 Da. Macfarlane's work has had a significant impact on many fields, especially biological research. Using the techniques he pioneered, biochemists have been able to obtain mass spectra of moderately sized proteins and other large biomolecules directly. Furthermore, and perhaps most importantly, his work established that large molecules could be desorbed from a target and recorded intact after passing through a mass analyzer. Macfarlane's research has inspired other workers in this area, leading to new discoveries and innovations (e.g., FAB and LD) that make it possible to record molecular masses as high as 300 000 Da. Nominations are now being solicited for next year's award, which will be presented at the 39th annual ASMS Conference in Nashville, TN, in May 1991. For more information, contact the ASMS Office, P.O. Box 1508, East Lansing, MI 48826 (517) 337-2548. Deadline for submission is October 30. Louise Voress
Supercritical Fluid G@3 Extraction Chromatography Monday-Thursday December 1 0 - 1 3 , 1 9 9 0 V i r g i n i a Tech Blacksburg, V A
An excellent course for those in need of practical lab experience in SFC/SFE Mow You'll Benefit from Ikfa Cowrie • Learn state-of-the-art techniques for performing SFE and SFC • Gain hands-on experience working with column detectors, chromatographs, and extractive devices • Use SFC to solve unique separation problems • Couple SFE with SFC • Interpret SFC data • Learn ways to interface chromatography with detection devices • and m u c h m o r e ! Course Instructor lorry T. Taylor, Profwor of Chembtry, Virginia Tech For m o r e information, call 202872-4508, extension 2103, or T O L L FREE 1-800-227-5558. Or use the coupon below to request a free descriptive brochure on this dynamic course. American Chemical Society Dept. of Continuing Education Meeting Code VPI1210 1155 16th St., N.W. Washington, D C 20036 Please send m e a brochure on the ACS Short Course, Supercritical Fluid Extraction/ Chromatography, to be held December 10-13, 1990, at Virginia Tech, Blacksburg, VA. Name Title Organization Address City, State, ZIP
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