Fig. 1. Overview of shotgun proteomics.

1) Sample proteins are digested into peptides using enzymes such as trypsin. Resulting peptide mixtures are optionally processed to capture a particular class of peptides (e.g. phosphorylated peptides), and then separated using a liquid chromatography (LC) system coupled online to a mass spectrometer. 2) Peptides are subjected to tandem mass spectrometry (MS/MS) analysis that results in the acquisition of MS/MS spectra. 3) The correct assignment of MS/MS spectra to peptide sequences is the first step in proteomic data processing.