Deep proteome coverage based on ribosome profiling aids mass spectrometry-based protein and peptide discovery and provides evidence of alternative translation products and near-cognate translation initiation events.

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Recently, a strategy, termed ribosome profiling (or RIBO-seq), based on deep sequencing of ribosome-protected mRNA fragments, indirectly monitoring protein synthesis, has been described. We devised a proteogenomic approach constructing a custom protein sequence search space, built from both Swiss-Prot- and RIBO-seq-derived translation products, applicable for MS/MS spectrum identification. To record the impact of using the constructed deep proteome database, we performed two alternative MS-based proteomic strategies as follows: (i) a regular shotgun proteomic and (ii) an N-terminal combined fractional diagonal chromatography (COFRADIC) approach. Although the former technique gives an overall assessment on the protein and peptide level, the latter technique, specifically enabling the isolation of N-terminal peptides, is very appropriate in validating the RIBO-seq-derived (alternative) translation initiation site profile. We demonstrate that this proteogenomic approach increases the overall protein identification rate 2.5% (e.g. new protein products, new protein splice variants, single nucleotide polymorphism variant proteins, and N-terminally extended forms of known proteins) as compared with only searching UniProtKB-SwissProt. Furthermore, using this custom database, identification of N-terminal COFRADIC data resulted in detection of 16 alternative start sites giving rise to N-terminally extended protein variants besides the identification of four translated upstream ORFs. Notably, the characterization of these new translation products revealed the use of multiple near-cognate (non-AUG) start codons. As deep sequencing techniques are becoming more standard, less expensive, and widespread, we anticipate that mRNA sequencing and especially custom-tailored RIBO-seq will become indispensable in the MS-based protein or peptide identification process. 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