Initiation of protein translation is a well studied fundamental process, albeit high-throughput and more comprehensive determination of the exact translation initiation sites (TIS) was only recently made possible following the introduction of positional proteomics techniques that target protein N-termini. Precise translation initiation is of crucial importance, as truncated or extended proteins might fold, function and locate erroneously. Still, as already shown for some proteins, alternative translation initiation can also serve as a regulatory mechanism. By applying N-terminal COFRADIC (combined fractional diagonal chromatography), we here isolated N-terminal peptides of a S. cerevisiae proteome and analyzed both annotated and alternative TIS. We analyzed this N-terminome of S. cerevisiae which resulted in the identification of 650 unique N-terminal peptides corresponding to database annotated TIS. Furthermore, 56 unique Nα-acetylated peptides were identified that suggest alternative TIS (MS/MS-based), while MS-based evidence of Nα-acetylation led to an additional 33 such peptides. To improve the overall sensitivity of the analysis, we also included the 5′ UTR (untranslated region) in-frame translations together with the yeast protein sequences in UniProtKB/Swiss-Prot. To ensure the quality of the individual peptide identifications, peptide-to-spectrum-matches were only accepted at a 99% probability threshold, and were subsequently analysed in detail by the Peptizer tool to automatically ascertain their compliance with several expert criteria. Furthermore, we have also identified 60 MS/MS-based and 117 MS-based Nα-acetylated peptides that point to Nα-acetylation as a post-translational modification since these peptides did not start, nor were preceded (in their corresponding protein sequence) by a methionine residue. Next, we evaluated consensus sequence features of nucleic acids and amino acids across each of these groups of peptides and evaluated the results in the context of publicly available data. Taken together, we present a list of 706 annotated and alternative TIS for yeast proteins and found that under normal growth conditions, alternative TIS might (co-)occur in S. cerevisiae in roughly one tenth of all proteins. Furthermore, we found that the nucleic acid and amino acid features proximate to these alternative TIS favour either guanine or adenine nucleotides following the start codon, or acidic amino acids following the initiator methionine. Finally, we also observed an unexpected high number of Nα-acetylated peptides that could not be related to TIS and therefore suggest events of post-translational Nα-acetylation.
Helsens K, Van Damme P, Degroeve S, Martens L, Arnesen T, Vandekerckhove J, Gevaert K. Bioinformatics analysis of a Saccharomyces cerevisiae N-terminal proteome provides evidence of alternative translation initiation and post-translational N-terminal acetylation. J Proteome Res. 2011 May 30. [Epub ahead of print]