Protein activity and turnover is tightly and dynamically regulated in living cells. Whereas the three-dimensional protein structure is predominantly determined by the amino acid sequence, posttranslational modification (PTM) of proteins modulates their molecular function and the spatial-temporal distribution in cells and tissues. Most PTMs can be detected by protein andpeptide analysis by mass spectrometry (MS), either as a mass increment or a mass deficit relative to the nascent unmodified protein. Tandem mass spectrometry (MS/MS) provides a series of analytical features that are highly useful for the characterization of modified proteins via amino acid sequencing and specific detection of posttranslationally modified amino acid residues. Large-scale, quantitative analysis of proteins by MS/MS is beginning to reveal novel patterns and functions of PTMs in cellular signaling networks and bio-molecular structures.

Introduction

Determination of posttranslational modifications (PTM) of proteins is fundamental in elucidation of the intricate processes that govern cellular events, like cell division, growth, and differentiation. The term PTM denotes changes in the polypeptide chain as a result of either the addition or removal of distinct chemical moieties to amino acid residues, proteolytic processing of the protein termini, or the introduction of covalent crosslinks between domains of the protein. PTMs are involved in most cellular processes including the maintenance of protein structure and integrity, regulation of metabolism and defense processes, and in cellular recognition events and morphology changes. Analysis of PTMs presents a number of challenges to protein and proteomics researchers, and efficient and sensitive methods for detection of PTMs are required. Traditionally, PTMs have been identified by Edman degradation, amino acid analysis, isotopic labeling, or immunochemistry. Within recent years, mass spectrometry (MS) has proven to be extremely useful in PTM discovery. The presence of covalent modifications in proteins affects the molecular weight of the modified amino acids, and the mass increment or deficit can be detected by MS (Table 1). MS has several advantages for characterization of PTMs, including (i) very high sensitivity; (ii) ability to identify the site of PTM; (iii) discovery of novel PTMs; (iv) capability to identify PTMs in complex mixtures of proteins; and finally (v) the ability to quantify the relative changes in PTM occupancy at distinct sites. None of the other techniques provide all these features. In this review we describe the utility of tandem mass spectrometry (MS/MS) for the determination of PTMs and provide selected examples of recent modification-specific proteomic studies.

Table 1. Mass Values for Some Posttranslational Modifications and Frequently Observed Neutral Losses and Diagnostic Ion Signals (Click to enlarge)

MS in Proteomics

MS is now widely used in protein biochemistry and in proteomics for the identification and characterization of proteins in cell lysates, isolated organelles, or purified multisubunit complexes (1,2,3). Protein separation technologies based on centrifugation, electrophoresis, or chromatographic methods are readily interfaced to MS in an online or off-line fashion. Proteins are then converted into peptides by treatment with sequence-specific proteases or chemical reagents, since peptides are more amenable to MS and MS/MS analysis than intact proteins.

The availability of robust and sensitive matrix-assisted laser desorption/ionization MS (MALDI-MS) (4) and electrospray ionization MS (ESI-MS) (5) instruments makes advanced MS technology accessible to molecular cell biologists, biochemists, and proteomics researchers. MS using either MALDI or ESI as the ionization method enables accurate mass determination of peptides. Peptide mass fingerprinting by MALDI-MS and subsequent sequence database searching is widely used for identification of proteins that are isolated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) or by two-dimensional gel electrophoresis (2DE) gels. In most studies it is also desirable to obtain amino acid sequence information, because the accurately determined molecular mass in combination with a partial amino acid sequence of a peptide are very specific probes for protein identification (1,6). Furthermore, mass analysis and amino acid sequencing by MS/MS may reveal the presence of PTMs at individual amino acid residues in proteins (3,7).

Peptide Sequencing by MS/MS

The tandem mass spectrometer provides the means for amino acid sequencing of peptides. It enables gas-phase isolation of individual peptide ion species followed by collision-induced dissociation (CID) and detection of the resultant amino acid sequence specific fragment ions (Figure 1). The MS/MS experiment consists of several stages of mass analysis and ion manipulation. First, the masses of the sample analytes are determined in a MS survey scan (first MS experiment). Second, the peptide ion of interest is isolated via its mass-to-charge ratio (m/z) value (i.e., by filtering away other ion species that have a different m/z value). Third, the selected peptide ion species is activated (e.g., by collisions with an inert gas such as Argon that imparts internal energy into the ions and thereby induces their fragmentation). Last, the m/z values of the fragment ions are determined (second MS experiment). The most labile bonds in peptides are generally the backbone amide bonds, leading to breakage of the peptide backbone in between amino acids. Hence, tandem mass spectra of peptides contain a series of sequence-revealing fragment ions (8). The fragment ion signals reflect the amino acid sequence as read from either the N-terminal (b-ion series) or the C-terminal (y-ion series) direction (9). The identities of the individual amino acids are revealed by the mass differences between the signals in b-ion series or y-ion series (Figure 1).