N-Terminal-oriented Proteogenomics of the Marine Bacterium Roseobacter Denitrificans Och114 using N-Succinimidyloxycarbonylmethyl)tris(2,4,6-trimethoxyphenyl)phosphonium bromide (TMPP) Labeling and Diagonal Chromatography

摘要: Given the ease of whole genome sequencing with next-generation sequencers, structural and functional gene annotation is now purely based on automated prediction. However, errors in structure are frequent, correct determination start codons being one main concerns. Here, we combine protein N termini derivatization using (N-Succinimidyloxycarbonylmethyl)tris(2,4,6-trimethoxyphenyl)phosphonium bromide (TMPP Ac-OSu) as a labeling reagent COmbined FRActional DIagonal Chromatography (COFRADIC) sorting method to enrich labeled N-terminal peptides for mass spectrometry detection. Protein digestion was performed parallel three proteases obtain reliable automatic validation termini. The analysis these enriched fractions by high-resolution tandem allowed refinement 534 proteins model marine bacterium Roseobacter denitrificans OCh114. This study especially efficient regarding analytical time. From validated termini, 480 confirmed existing annotations, 41 highlighted erroneous codon five revealed totally new mis-annotated genes; data also suggested existence multiple sites eight different genes, result that challenges current view translation initiation. Finally, identified several which classical homology-driven inconsistent, questioning validity pipelines emphasizing need complementary proteomic data. All have been deposited ProteomeXchange identifier PXD000337.