Figure 1: Shotgun proteomics for a proteogenomic approach: most common strategy and current tools. The most classical approach in shotgun proteomics consists in i) fractionation of proteins upon their molecular weights by 1D SDS-PAGE, prior to ii) trypsin proteolysis of the protein fractions (polyacrylamide bands), and iii) peptide identification by nanoLC-MS/MS with high throughput hybrid mass spectrometers. MS/ MS data are then assigned to peptide sequences by specific searches against publicly available databases or homemade databases comprising all the possible protein sequences. For proteogenomics, the database is made of a sixframe translation of the nucleic acid sequence. Such approach typically results in assignment of tens of thousands MS/MS spectra and identification of hundreds of proteins with two or more non-redundant peptides.