altern - Bioinformatics analysis of a Saccharomyces cerevisiae N-terminal proteome provides evidence of alternative translation initiation and post-translational N-terminal acetylation

S.cerevisiae proteomes were prepared for LC-MS/MS analysis using an Ultimate 3000 HPLC system (Dionex, Amsterdam, The Netherlands) in-line connected to a LTQ Orbitrap XL mass spectrometer (Thermo Electron, Bremen, Germany). Mascot server version 2.2 from Matrix Science was then used to identify the MS/MS spectra in the S. cerevisiae content of UniProtKB/Swiss-Prot (version 15.10) concatenated with the 5-UTR peptide centric database derived from SGD. A shuffled version of this concatenated database was created to estimate the false discovery rate in the results at 0.64%. The precursor ion tolerance was set to 10 ppm and the fragment ion tolerance was set to 0.5 Da. Semi-specific Arg-C/P was used as enzyme specificity and no missed cleavages were allowed. The fixed modifications were 13C2D3-acetylation (+47 Da) on Lys, carbamidomethylation (+57 Da) on Cys and oxidation (+16 Da) on Met, and the variable modifications were acetylation (+42 Da) and 13C2D3-acetylation (+47 Da) on the N-terminus. N-terminal propionylation (+56 Da) was set as an additional variable modification in a parallel search for N-terminal propionylation. The charge state was set to allow single, double and triple charged peptides. All peptide identifications were subsequently processed, stored and managed by ms-lims.