Trypsin is the most widely used protease, cleaving proteins with high specificity and generating peptides 7–20 amino acids long with a strong C-terminal charge (1) , ideal for mass spectrometry analysis. However, trypsin has certain limitations. Tightly folded proteins resist trypsin digestion, and inadequate distribution of trypsin cleavage sites in certain proteins or protein domains generates peptides that are too long or too short for mass spectrometry analysis. Membrane proteins often exhibit both resistance to trypsin and few trypsin cleavage sites, requiring alternative approaches when preparing for mass spectrometry (2) (3) . Post-translational modifications (PTMs) present yet another challenge because glycans often limit trypsin access to cleavage sites whereas acetylation or di- and trimethylation of lysine and arginine residues make them resistant to trypsin digestion (4) (5) (6).
We provide several alternative proteases that can be used when trypsin is not informative. Lys-C protease is active under denaturing conditions, offering the means to overcome proteolytic resistance of tightly folded proteins. Chymotrypsin preferentially cleaves at aromatic and other hydrophobic residues and, therefore, can digest hydrophobic proteins. Asp-N and Glu-C proteases add flexibility when choosing protein cleavage sites, providing a solution when trypsin does not generate peptides within the optimal size range or PTMs interfere with trypsin proteolysis.
However, mass spectrometry analysis of proteins digested using Lys-C, Asp-N, Glu-C, chymotrypsin and trypsin rarely produce complete protein coverage. Incomplete sequence coverage decreases the number of PTMs available for analysis and diminishes the ability to distinguish between proteins with a high degree of sequence similarity. Here we show that the proteases Arg-C, elastase, thermolysin and pepsin address these issues by increasing protein sequence coverage or digesting under alternative conditions such as higher temperature or lower pH. We demonstrate the advantages of these proteases using various model proteins or protein mixtures, including a yeast total protein extract, a PTM-rich human histone H4, phosphorylase B and bacteriorhodopsin.