System-wide proteomic identification of protease cleavage products by terminal amine isotopic labeling of substrates

The sequence and nature of all the protein amino-termini (N-termini) within the proteome (the N-terminome) provides valuable functional annotation, since translation start sites, N-terminal isoforms, modifications and truncations determine the cellular localization, activity and fate of most proteins (1). As ~ 85% of eukaryotic proteins have an acetylated N-terminus (2) and all proteins undergo proteolysis (3), these are not only two of the most ubiquitous, but also two of the most important post-translational modifications (4,5). The protein amino-terminus is susceptible to amino-terminal peptidase processing, modification of the alpha-amino group, and side-chain specific changes that can target a protein for ubiquitination and degradation or protect it from rapid turnover and so determines its half-life (1). In addition to constitutive proteolysis, regulated processing of protein amino termini can irreversibly change the protein activity or properties (6-8) but the extant to which proteolysis sculpts the proteome is unknown (4). Hence, it is important to determine the cleavage site within each protease substrate, since the biological activity of the cleavage products is commonly determined by the precise fragmentation pattern.