Solution Protein Digest

Abstract

INTRODUCTIONThis protocol describes the trypsin digestion of proteins in solution. Proteolytic digestion is critical for mass spectrometric sequencing because it generates peptides that have molecular weights within the mass range of the mass spectrometer. For tandem mass spectrometry, the digestion is typically performed using the protease trypsin, which cleaves at the carboxy-terminal side of lysine (K) and arginine (R) residues with the exception of K-P and R-P sites. There are numerous variations for digestion of proteins in solution. Because trypsin is a rather robust enzyme, tryptic digestions can be performed under various denaturing conditions (4 M urea, 2 M guanidine-HCl, 0.1% SDS, and >10% acetonitrile). Solubilization of proteins in 8 M urea and then dilution to 2-4 M urea before adding trypsin is commonly used to digest proteins that are difficult to solubilize. For most digestion protocols, including the protocol described in this article, cysteine residues are reduced and alkylated prior to digestion. The reduction and alkylation of disulfide bonds help to denature proteins, making their proteolytic sites more accessible to proteolysis. The The modified trypsin (TPCK-treated) used in this protocol is a serine endopeptidase prepared by treating trypsin with L-(tosylamido-2-phenyl) ethyl chloromethyl ketone (TPCK) to inactivate any remaining chymotryptic activity. TPCK acetylates the ϵ-amino groups of lysine residues to limit autolysis. Modified trypsin cleaves at K-P and R-P bonds at a much slower rate than other amino acid residues.