Mass Spectrometry

Although 2D-PAGE capabilities continue to advance, the field has increasingly turned to direct mass spectrometric analysis of complex mixtures (Fig. 3), a significant break-

Fig. 4. Schematic of tandem MS.

through that has enabled many ofthe accomplishments ofproteomics analysis. The experimental origins of modern MS date back more than 100 yr, when J. J. Thomson noted that the parabolic trajectory exhibited by ions moving through electric fields was proportional to their "mass-to-charge (m/z)" values. The first instrument, a parabola spectrograph in 1912, was developed from this observation. Later advances in vacuum technology and electronics led to smaller and more sophisticated instrumentation, with Nobel prizes being awarded to Wolfgang Paul (1999, physics) and John Fenn (2000, chemistry) for their contributions on the quadrupole mass analyzer/ion trap and electrospray ionization (ESI), respectively.

MS instrumentation is unrivaled in its ability to offer several layers of complementary information, having benefited tremendously from whole-genome analysis and the genomics revolution. MS can provide mass measurements of intact proteins far more accurately than can be obtained by gel electrophoresis. MS can also provide accurate mass detection of peptides from proteolytic digests of complex mixtures, with even higher sensitivity and mass accuracy than for detection of whole proteins. The set ofpeptide mass measurements can be searched against databases, frequently to obtain definitive identification of the parent proteins of interest. Favorably compared against other proteomics technologies, MS is highly sensitive (MS/MS technology, described subsequently, can detect and characterize attomole quantities of peptides) and amenable to automation and thus promotes high-throughput processing. More important, MS has a wide range of applicability and can not only detect proteins but also characterize any posttranslational modifications.

Mass spectrometers are composed of modular elements, which include an ion source, a mass analyzer, and a detector/recorder (see Fig. 4). Different types of MS instruments are classified according to the ionization source and mass analyzer employed, but all process samples as gas-phase ions, whose movements within an electromagnetic field can be precisely measured. An ion source generates these gas-phase ions from the analyte through a variety of available techniques, from either the solid state by matrix-assisted laser desorption ionization (MALDI), or directly from the liquid phase by ESI. Modern ESI systems are coupled to antecedent chromatography at low flow rates to fractionate complex peptide mixtures. Gas-phase ions are subsequently directed into the mass analyzer, which resolves the peptides based on their m/z ratio. Examples of commonly used mass analyzers include the quadrupole mass filter, ion trap mass analyzer, and time-of-flight (TOF) mass analyzer.

Finally, the detector detects the ions via an electronic multiplier, recording ion intensity vs m/z value to create the resulting MS spectra. Each protein or set of proteins will yield a unique set of distinct peptides, termed a peptide mass fingerprint. The set of peptide masses obtained by MS can be compared to the theoretical mass fingerprints of proteins obtained by in silico cleavage ofknown sequences in searchable databases. However, unambiguous protein identification often requires additional protein sequence information.

In MS/MS, a single m/z species from the mixture of peptide ions is selected for further analysis. This ion is subjected to collision-induced dissociation, which generates fragment ions from the parent peptide ion. The fragment ions are then analyzed on the basis of their m/z in a second stage of mass analysis, resulting in a product ion spectrum. The information contained in this tandem MS spectrum permits the amino sequence of the peptide to be deduced. Obtaining an accurate mass or the sequence of a peptide allows one to score its identity against a database of known protein sequences. Most peptide sequences of approximately six or more amino acids in length are uniquely represented in the proteome of an organism and will map to a single gene product. When the sequence corresponds to a common motif, as found in many structural or binding sites, additional information is necessary. A presumed match is corroborated if additional peptide sequences from the same protein are identified.

Again, simplification ofthe mixture allows data to be collected on the greatest number of components. It is possible to separate the intact proteins first and then cleave them into peptides. It is also possible to cleave the proteins into peptides first and then separate the peptides prior to analysis. As demonstrated in Fig. 4, a combined approach is now generally considered necessary. The peptides are then analyzed as we have outlined, by either TOF instruments, to measure the masses and identify proteins by fingerprinting or, preferably, by ESI-MS/MS, to obtain direct sequence data.

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