Mass Spectrometry and Proteomics

Courtesy of Thermo Electron For years, mass spectrometry has been de rigeur in chemistry labs. Recently, though, it has become a mainstay of proteomics research, too. Two years ago, investment bank UBS Warburg identified proteomics as the fastest- growing application of mass spectrometry, a prediction borne out at this year's American Society for Mass Spectrometry (ASMS) annual meeting, where more than 1,100 of 2,227 presentations discussed some aspect of proteomics. It took a confluence of trends to effect this change. First, in the late 1980s, two new ionization methods, MALDI (matrix-assisted laser desorption ionization) and ESI (electrospray ionization), allowed routine mass analysis of large biomolecules. Second, biologists began to recognize that proteins, rather than genes, describe what happens inside cells. Finally, swelling DNA sequence databases, coupled with computers able to search them rapidly, made it possible to identify novel proteins, and the genes that encode them, from MS-derived peptide sequences. Today, proteomicists use MS to identify and characterize proteins (elucidating their primary and tertiary structure, relative abundance, and post-translational modifications, for instance), and to dissect interactions among and between proteins and small molecules. Biologists who once could spend years trying to describe the phosphorylation pattern of a particular protein can get an answer in just weeks today. And the work is being done with ever improving sensitivity and accuracy. Modern instruments can sift through complex peptide mixtures to find one in particular. "It's not unusual for us to look for one molecule in 10,000," states Chris Becker, director of chemistry at SurroMed in Mountain View, Calif. Yet instrument developers and users are not resting on their laurels. At the recent ASMS meeting, researchers unveiled new hardware, reagents, and approaches, all addressing some previously unmet need. Other investigators demonstrated how they used MS to solve problems in the clinic, giving a possible hint to the field's future in diagnostics work. WHAT'S IN A NAME? The alphabet soup of MS nomenclature confounds many nonexperts. Most confusion arises when terms describing the three major components of an MS instrument--ionization method, analyzer, and detector--are mixed and matched. MALDI and ESI, the preferred ionization methods for proteins, are recognized as the enabling MS technologies for proteomics. In MALDI, laser light vaporizes proteins embedded in a solid matrix of photon-absorbing material. Electrospray also achieves free gas-phase ions, but from solution phase, which is a powerful capability when coupled with liquid chromatography (LC). Older ionization methods such as electron impact and chemical ionization work primarily with volatile, low-molecular-weight molecules. Evan Williams, a chemistry professor at the University of California, Berkeley, notes how MS evolved stepwise after adoption of novel ionization methods, including laser desorption in the 1960s, plasma desorption in the 1970s, and fast-ion bombardment in the 1970s and 1980s. "But MALDI and ESI changed everything by bringing molecules with molecular weights in the hundreds of thousands or even millions into the gas phase." ESI and MALDI ionizations allow mass analysis of proteins, polymers, and even noncovalent supramolecular species like protein-protein, protein-DNA, and protein-drug complexes. Few biologists use MS for masses above 100,000 daltons or so, the "frontier of MS," says Mark Bier, professor of chemistry at Carnegie Mellon University in Pittsburgh, Pa. "Although MALDI and ESI are capable of analyzing much higher masses, their popularity is based on the ability to fly and mass-analyze biomolecules of between 500 and 100,000 daltons," he says. Among mass analyzers, time-of-flight (TOF), quadrupole, quadrupole ion traps (QIT), Fourier-transform ion traps (FT/IT), and more recently Fourier-transform ion cyclotron resonance (FT/ICR) spectrometers (and combinations thereof) are most common in proteomics. FTMS' exquisite mass-resolving capability makes it ideal for analyzing large molecules, mixtures of proteins or peptides, and relatively small posttranslational modifications. At ASMS 2003, Pfizer's George L. Perkins demonstrated that FTMS is about four times as accurate at resolving protein fragment masses as QTOF, and about 11 times more accurate than magnetic sector analyzers. Such precision has its price, however: FTMS requires very high-field magnets, which must be kept cold with liquid hydrogen and liquid nitrogen. Also, says Bier, FT instruments are expensive. "But that will change as the technique becomes more popular." PROMOTING TOGETHERNESS Another source of confusion in MS discussions is use of the term "tandem." Single-stage MS experiments use one mass analysis step. But tandem MS employs multiple steps to probe primary structures and daughter ion fragments. Some tandem instrument setups employ discrete analyzers arranged in physical sequence (e.g., two quadrupoles [double quadrupole] or a quadrupole and a TOF [QTOF]) and are referred to as tandem-in-space. Ion traps also perform tandem analysis, but use only one physical analyzer and are called tandem-in-time. Such "virtual" tandem analyzers concentrate ions, fragment them, and analyze in successive iterations to generate an MS fingerprint, all in one physical instrument. Rohan Thakur, engineering program manager for MS instrument-maker Thermo Electron, compares tandem-in-space MS to running a traditional footrace, and tandem-in-time to running in place. Since every stage of a tandem-in-space tandem MS involves material loss, the method's practical limit is MS3. Ion traps, which don't waste ions or ion products, provide up to MS10; the only limit is the number of ions stored at each isolation step. One common application of tandem MS is peptide sequencing. Top-down mass-based sequencing uses MSn, often with ion trap or FT/ICR spectrometers, to ionize proteins and analyze fragments down to amino acid ions. Bottom-up sequencing involves enzymatically digesting proteins, identifying mass ions, and comparing the results with sequence databases. "The top-down approach is making a comeback," says Mark Bier. "It's typically used to identify residues containing specific posttranslational modifications such as glycosylations and phosphorylation." Courtesy of Thermo Electron TANDEM-IN-TIME: Ion traps, like these from Thermo Electron, perform tandem mass spec functions in a single instrument. CLINICAL RELEVANCE With so many technical improvements unveiled at ASMS (see sidebar below), it's easy to forget MS's clinical value. By characterizing complex biomolecular mixtures, MS can help researchers identify new disease diagnostics. Steve Martin, Senior Director of Discovery Proteomics at Applied Biosystems, says the marriage of proteomics and clinical research is the hottest trend in MS, and ASMS 2003 featured several sessions on the topic. Lawrence Fischer from the Hospital for Sick Children, Toronto, compared tandem MS with standard instrumental analysis of physiologic amino acids and found the former to be much faster, more sensitive, and more user-friendly. Similarly, Mark Hows of GlaxoSmithKline used derivatization followed by MS/MS to assay amino acids in the central nervous system. Andrea Mardegan and coworkers at the University of Padua, Italy, demonstrated identification of 3b-hydroxysteroid-d5-oxidoreductase deficiency, a rare metabolic disorder, from ESI-MS/MS analysis of urine. Bethesda, Md.-based Correlogic Systems and Ithica, NY-based Advion BioSciences reported on an MS protein profiling technique for diagnosing ovarian cancer. Beginning with blood samples from 57 normal and 97 cancer samples, researchers used Correlogic's Proteome Quest™ heuristic bioinformatics algorithm software and ESI-TOF analysis to train their system to recognize samples from patients with cancer. An additional 38 control and 83 cancer samples were randomly, blindly analyzed. This technique identified samples from patients with cancer with a sensitivity of 87% and a specificity of 84%. Meanwhile, John Marshal of Synx Pharma, Toronto, reported in the Journal of Proteome Research (doi:10.1021/pr0300031) on a MALDI-TOF technique that distinguishes patients who had recent heart attacks from control subjects. Marshal examined peptides resulting from two cleavage cascades. The first, involving complement C3 alpha, results in a peptide that is further degraded after myocardial infarction by aminopeptidases. The second family, also hydrolyzed by aminopeptidase, arises from a peptide A fragment from fibrinogen. Angelo DePalma (adp@tellurian.net) is a freelance writer in Newton, NJ. HIGHLIGHTS FROM ASMS 2003 Courtesy of Bruker Daltonics Instrumentation: Jerry Moore from Argonne National Laboratory presented data on tunable vacuum ultraviolet ionization (VUVI), which detects mutant DNA and surface-catalyzed reactions. The key is tunable VUV, made possible by Argonne's free electron laser--one of only two in existence--which softly ionizes large molecules with minimal fragmentation and at high efficiency. Moore claims that tunable VUV will detect low-abundance mutant DNA against a backdrop of wild-type genes. Purdue researcher Amy Tabert described a high-throughput miniature cylindrical ion trap array (CITA) MS instrument, jointly developed with Thermo Electron of Bremen, Germany. CITA's claims to fame are multiplexing and high throughput, both of great interest for deciphering protein mixtures in bioprocess monitoring and metabolomics. The new technique can, for example, analyze four different proteins simultaneously, or one protein in each of four channels. Ion mobility TOF, a multidimensional method developed at Beyond Genomics in Waltham, Mass., separates ions by their mobility through buffer gas as well as by their mass/charge ratio. The technique exploits differences in molecular movement between drift time, which occurs over milliseconds, and TOF, which happens a thousand times faster. Thus each group of ions exiting the drift tube may be analyzed several times to give a TOF spectrum. Data presented at ASMS 2003 showed the technique to be sensitive, too. At 25 femtomole of peptide detected, ion mobility TOF operates near the lower limit of conventional MS proteomics. A linear ion trap MS invented by Thermo Electron's John Syka and Mark Bier (who used to work at the company) is now being commercialized at Thermo. Bier's instrument uses a larger-than-normal, elongated ion trap that concentrates more electrons than conventional instruments, without requiring an increase in charge density. Bier claims his design improves signal-to-noise, sensitivity, detection limit, and dynamic range. Kermit Murray of Louisiana State University presented data on a microfluidic interface for MALDI MS, a new way to get small samples into a spectrometer. Murray's device, based on a rotating ball inlet, uses a mechanically driven steel ball to deliver analyte to the MS vacuum chamber. Membrane Proteins: As many as one-third of all proteins, and as much as 70% of clinically relevant proteins, are membrane-associated, which makes them difficult to study through conventional biochemistry. Julian Whitelegge of University of California, Los Angeles, described a two-dimensional liquid chromatography method that extends LC/MS to complex membrane proteins. The first LC dimension delivers intact complexes while the second, run under mildly denaturing conditions, produces intact, uncomplexed proteins which are analyzed for cleavage patterns and fingerprinting. "The problem with one-dimensional separation is it only affords so much spatial resolution," Whitelegge told The Scientist. "For intact proteins with our LC-MS methods, mass resolution is around 100. The idea of the second dimension, inserted up front, is to break down the mixture complexity." Similarly Sonja Hess of the National Institute for Diabetes and Digestive Diseases discussed LC-MS and LC-MS/MS techniques that characterize fimbrial membrane proteins on Gram-negative and Gram-positive bacteria. Studying fimbrial proteins, which are implicated in bacterial virulence, has been hampered by the structures' hydrophobicity. By solubilizing fimbriae with 5% hexafluoroisopropanol in acetonitrile, Hess characterized enough of the solubilized proteins by LC-ESI MS/MS to keep anti-infective drug discovery groups active for some time. Stable Labels: ASMS 2003 featured several sessions on stable-label MS. In such an experiment, two samples that differ in isotopic enrichment are mixed. Because the molecules serve as their own internal standard within the MS instrument, resulting peak ratios directly measure relative expression. Christine Wu of The Scripps Research Institute in La Jolla Calif., presented a method for metabolically labeling selected rat tissues with 15N at greater than 90% incorporation. Wu obtained differential protein profiles for 369 rat liver proteins by comparing homogenized liver from labeled and unlabeled rats. She described her work as the "first nongel" quantitative proteomic tissue analysis. Gavin Reid from the Ludwig Institute for Cancer Research, Victoria, Australia, showcased a new technique, SELECT (selective extraction of labeled entities by charge derivatization and tandem MS), for quantifying differential protein expression. SELECT addresses a nagging problem in transferring knowledge from genomics to proteomics, namely that gene expression predicts protein abundance poorly. Reid's MS/MS method uses peptide derivatization and differential isotope labeling to identify, without purification, peptides in complex mixtures containing specific residues. Reid claims a 100-fold sensitivity improvement over conventional stable-label methods by using specific, fixed-charge amino acid derivatization that results in predictable fragmentation. This overcomes limitations of differential labeling, for example when low-abundance labeled materials overlap with higher-abundance components or when labeled molecules cannot be discerned from noise. function sendData() { document.frm.pathName.value = location.pathname; result = false if (document.frm.score[0].checked) result = true; if (document.frm.score[1].checked) result = true; if (document.frm.score[2].checked) result = true; if (document.frm.score[3].checked) result = true; if (document.frm.score[4].checked) result = true; if (!result) alert("Please select") return result; } .myradio{background-color : #94bee5} Please indicate on a 1 - 5 scale how strongly you would recommend this article to your colleagues? Not recommended 1 2 3 4 5 Highly recommended Please register your vote

Mass Spectrometry and Proteomics

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