Trypsin, the most widely used protease in mass spectrometry analysis, cleaves proteins in a highly specific manner. However, tightly-folded proteins can resist trypsin digestion. Post-translational modifications (PTMs) present a different challenge for trypsin because glycans often limit trypsin access to cleavage sites, and acetylation makes lysine and arginine residues resistant to trypsin digestion. We offer several alternative proteases that complement standard trypsin digestions and provide enhanced data.
Digestion with an alternative protease individually or in combination with other proteases creates a unique peptide map that may include sequences not seen with trypsin digestion alone (Figures 1 and 2).
Figure 3. Histone H4 post-translational modifications identified in trypsin and Arg-C digests.
Thermolysin and pepsin are unique because they tolerate conditions that can be used to denature difficult to digest and tightly folded proteins, high temperatures and low pH, respectively. Because they remain active under these conditions, these proteases can cleave previously inaccessible sites.