Bottom-up proteomics focuses on the analysis of protein mixtures after enzymatic digestion of the proteins into peptides. The resulting complex mixture of peptides is analyzed by reverse-phase liquid chromatography (RP-LC) coupled to tandem mass spectrometry (MS/MS). Identifying peptides, and subsequently proteins, is completed by matching peptide fragment ion spectra to theoretical spectra generated from protein databases.
Trypsin is the most widely used protease in mass spectrometry sample preparation. It is a highly specific serine protease, which cleaves at the carboxylic side of lysine and arginine residues. Protein digestion with trypsin generates peptides of optimal sizes for mass spec analysis.
There are certain proteins and protein mixtures where trypsin digestion alone is not efficient enough. Examples include digestion of membrane proteins and analysis of histone post-translational modifications (PTMs). Digestion with an alternative protease, individually or in combination with trypsin, creates a unique peptide map that may include sequences not seen with trypsin digestion alone. Overlaying peptides obtained with alternative proteases and trypsin increases protein coverage and overall confidence in protein identification.
