Authors
Oded Kleifeld, Alain Doucet, Jayachandran N. Kizhakkedathu, and Christopher M. Overall
Summary
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 proteins1. As ~ 85% of eukaryotic proteins have an acetylated N-terminus2 and all proteins undergo proteolysis3, these are not only two of the most ubiquitous, but also two of the most important post-translational modifications4,5. 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-life1. In addition to constitutive proteolysis, regulated processing of protein amino termini can irreversibly change the protein activity or properties6-8 but the extant to which proteolysis sculpts the proteome is unknown4. 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.
With 569 members, proteases are the second largest enzyme class in man9 and are 5-10% of drug targets10. Crucial to linking a specific protease with a defined biological pathway and for drug development is determining the substrate repertoire, or substrate degradome11, of a protease since this can generate hypotheses on its role and provide biomarkers of drug efficacy. Yet, for around half of the proteases in man no substrates are known and for the other half, the substrate degradome is incompletely annotated3,11.Thus specific degradomics techniques are needed to rapidly identify and quantify the N-terminome in order to reveal the extent of proteolysis in a system, the functional state of key molecules, and to identify new substrates.
Positional proteomics approaches that isolate only the N-terminal peptides of proteins, the N-terminome12-16, have been proposed for sample simplification before mass spectrometric (MS) analysis and for proteome annotation, but coverage is often limited12-15,17,18 and aside from combined fractional diagonal chromatography (COFRADIC) most of these approaches were not reported for global protease cleavage site analysis. The main obstacles towards this latter goal are the identification the neo-N-termini of the substrates generated by specific proteolysis and to distinguish these not only from the natural N-termini, but also from N-termini generated by background proteolysis of the proteins in a sample and by trypsin digestion (internal tryptic peptides) in proteomic workflows3,19. Solving these problems requires innovative strategies to circumnavigate the very similar chemical properties of the primary amines of the lysine side chains and N-termini. Recent reports of different approaches to tackle this difficult task include lysine-specific blocking of intact proteins to expose only amino-termini for biotinylation followed by affinity capture20, specific labeling followed by in silico selection of protease generated neo-N-terminal peptides21, specific enzyme-mediated biotinylation of unblocked alpha-amine groups with enrichment for the biotinylated N-terminal peptides after one reaction16. Although these approaches represent a welcome step forward for studying protease-generated neo-N-terminal peptides, they are still limited in different aspects such as quantification20, coverage21 or are potentially biased16 and more importantly incapable of analysing naturally blocked N-terminal peptides. To date, COFRADIC is the only N-terminomics approach that provides, broad coverage and isotopic quantification and can be applied to study protease substrate degradomes as well as to completely annotate the N-terminome12,22-25. However COFRADIC is an expensive and time-consuming procedure involving multiple and complicated enzymatic and chemical steps, multiple HPLC fractionations and up to 150 MS/MS analyses per experiment25.
To overcome these problems we developed a new positional proteomics approach: Terminal Amine Isotopic Labeling of Substrates (TAILS)26. TAILS is a combined N-terminomics and protease substrate discovery degradomics platform for the simultaneous quantitative analysis of the N-terminome and proteolysis on a proteome-wide scale in one MS/MS analysis. By a three-day procedure with flexible labeling options, TAILS removes internal tryptic and C-terminal peptides to enrich for all forms of N-terminal peptides by negative selection. To overcome nonspecific peptide binding and the low capacity of derivatized chromatographic beads that in some techniques necessitates large sample amounts for analysis, we developed a novel class of dendritic polyglycerol aldehyde polymers optimized for efficient, high capacity tryptic peptide binding with virtually no non-specific interactions. Rather than deliberately excluding acetylated proteins13,16,20,21, TAILS provides wide coverage of all forms of naturally blocked N-terminal peptides and allows for their quantification through isotopic labeling of lysine side-chains. In addition to annotating the proteome we utilze these peptides to form a statistical classifier for the TAILS experiments to determine statistically valid isotope ratio cutoffs.
Further details
The protocol was published on Protocol Exchange on 7 March 2010. To see the entire protocol, click on the source link.
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