Meyer, J.G. et al. Mol. Cell. Proteomics doi:10.1074/mcp.M113.034710 (14 January 2014).

Trypsin has long been the workhorse protease for bottom-up proteomics, in which proteins are digested into peptides, the peptides are analyzed by mass spectrometry (MS), and the resulting spectra are identified by database searching. However, many peptides generated by trypsin digestion are of non-optimal length for MS analysis, meaning that important post-translational modifications can be missed. Several alternative proteases, such as LysC, are also in use, but like trypsin, most of these cleave at charged amino acids. Meyer et al. now report two new proteases for bottom-up proteomics that cleave at aliphatic side chains: wild-type alpha-lytic protease (WaLP) and an active site mutant, M190A alpha-lytic protease (MaLP). Meyer et al. combined proteomics data from separate digestions of the Saccharomyces pombe proteome with WaLP, MaLP, trypsin and LysC. This combination boosted sequence coverage of the proteome substantially compared to the coverage obtained with the use of trypsin alone and especially in hydrophobic transmembrane regions.