Methods for the proteomic identification of protease substrates

Abstract

Proteolysis is a key regulatory post-translational modification in diverse cellular processes including programed cell death, immune function, and development. Tracking proteolytic events has become a focus of researchers assessing the downstream consequences of protease activation. In this review we summarize unbiased methods for identifying protease substrates and tracking the extent of cleavage, a field termed 'degradomics'. These include one-dimensional and two-dimensional gel-based methods for identifying protease substrates, N-terminal peptide identification methods for simultaneously identifying substrates and cleavage sites, and approaches for the quantitation of cleavage events during endogenous proteolysis. Individual methods have identified more than 300 caspase-cleaved targets during apoptosis suggesting broad future applications for these technologies.

Figure 1. Gel-based methods for identifying protease substrates

A. 2-D DIGE: A control and proteolyzed lysate are labeled with fluorescent dyes, mixed, and analyzed by 2-D gel electrophoresis. Spots with unequal fluorescence ratios are picked as proteolyzed proteins for MS analysis. B. 2-D SDS-PAGE: A lysate is resolved by SDS-PAGE, proteolyzed, and electrophoresed perpendicular to the initial migration. Spots occurring below the diagonal are identified as substrates. C. PROTOMAP: Control and proteolyzed cell lysates are analyzed side-by-side via SDS-PAGE. The gel is cut into bands, trypsinized, and peptides are identified by LC-MS/MS. For each protein, peptides are analyzed by peptographs (analysis tools that display the sequence coverage and intensity in each band), revealing the approximate site(s) and extent of cleavage.

Figure 2. Methods for isolating N-terminal Peptides

A. Lysates are treated with chemical reagents or enzymes to modify protein amines. Subsequent trypsinization reveals N-terminal peptides with derivatized N-termini and internal peptides with free amines. B. In negative enrichment methods, internal peptides are removed by reaction with an amine-reactive reagent followed by chromatography or solid phase extraction. The remaining peptides are analyzed by LC-MS/MS. C. In positive enrichment methods, N-terminally labeled peptides are isolated by bead capture, and chemically or enzymatically released for analysis by LC-MS/MS.

Figure 3. Methods for relative peptide quantification

A. SILAC. Duplicate cell cultures are grown in media containing light/heavy amino acids, proteolyzed separately, mixed, and then the N-termini are isolated. Relative peptide intensities are determined by comparing MS intensities. B. MS-based peptide quantification. Cells are lysed, protelyzed, and their N-termini are isolated and derivatized with isotopically labeled reagents. The modified peptides are mixed and quantified based on MS intensities. C. MSMS-based peptide quantification. Isolated N-termini are labeled with isobaric iTRAQ reagents, each with distinct isotopic compositions, and mixed. At the MS level of analysis each peptide is identical. At the MS/MS level the isobaric tag fragments to reveal the relative intensities of the four samples.