Mass spectrometry of peptides and proteins
Introduction
Mass spectrometry and tandem mass spectrometry (MS/MS) experiments are major tools used in protein identification. Mass spectrometers measure the mass/charge ratio of analytes; for protein studies, this can include intact proteins and protein complexes [1], fragment ions produced by gas-phase activation of protein ions (top-down sequencing) [2], [3], [4], [5], [6], peptides produced by enzymatic or chemical digestion of proteins (mass mapping) [7], [8], and fragment ions produced by gas-phase activation of mass-selected peptide ions (bottom-up sequencing) [9]. The application of mass spectrometry and MS/MS to proteomics takes advantage of the vast and growing array of genome and protein data stored in databases. The information produced by the mass spectrometer, lists of peak intensities and mass-to-charge (m/z) values, can be manipulated and compared with lists generated from “theoretical” digestion of a protein or “theoretical” fragmentation of a peptide. Applications to analyze ever smaller quantities of sample are driving the development of more sensitive mass spectrometers, as well as low flow, high resolution separation technologies, to provide structural information on individual components in complex mixtures of thousands of proteins derived from biological samples. Protein identification by mass spectrometry requires an interplay between mass spectrometry instrumentation (how molecules are ionized, activated, and detected) and gas-phase peptide chemistry (which bonds are broken, at what rate, and how cleavage depends on factors such as peptide/protein charge state, size, composition, and sequence). This brief tutorial article provides an overview of peptide and protein fragmentation in mass spectrometers.
Section snippets
Instrumentation
A rich variety of different MS/MS instrument configurations (with different capabilities in terms of speed, ionization method, resolution, sensitivity, and mass/charge range) have been developed both in research laboratories and in the marketplace for application to proteomics. (For a tutorial on mass spectrometry instrumentation, refer to http://staging.mc.vanderbilt.edu/msrc/tutorials/ms/ms.htm.) This large number of instrument types has developed because no one instrument type has all of the
Top-down sequencing: protein fragmentation in the gas phase
An approach that involves direct protein sequencing in the gas phase is referred to as top-down sequencing and has been demonstrated [2] and developed [3], [4], [5] over the past decade. This approach is an alternative to the commonly used bottom-up sequencing (see next section). In the top-down approach, the protein sample is not subjected to enzymatic digestion, but instead transferred into the gas phase intact. Subsequent measurement of the protein molecular weight and fragmentation of the
Bottom-up sequencing: peptide fragmentation in the gas phase
The more popular approach to protein identification relies on peptide sequencing and is referred to as bottom-up sequencing. This approach requires accurate sequence analysis of the MS/MS spectra of the proteolytic fragments so that protein identification can be made and typically relies on algorithms for amino acid sequence assignments.
Fragmentation mechanisms and algorithm development
In the early 1990s, computer search algorithms for identifying proteins from peptide mass spectral data became available allowing for the high-throughput identification of unknown proteins [68], [69], [70], [71], [72]. Pioneering studies in this area utilized in-gel digestion protocols and began to establish databases of proteins expressed in human myocardial cells, melanoma cells, and yeast [73], [74]. The term “proteomics,” used to describe the sum of proteins expressed in a given cell type,
Concluding remarks
In the last 15 years, mass spectrometry applications have revolutionized analysis of proteins, moving from simple studies of purified proteins, blocked N-termini, modified peptides, and analysis of peptide synthesis reactions, to the current dizzying array of new methods and instruments, as well as inspiration for the new field of systems biology. Proteomics is now a multibillion-dollar enterprise. In the same time, we have shifted from an era where our understanding of protein and peptide
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