The use of trypsin in bottom-up proteomics may impose certain limits in the ability to grasp the full proteome. 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.
To overcome these problems, the proteomics community has begun to explore alternative proteases to complement trypsin. However, protocols, as well as expected results generated when using these alternative proteases have not been systematically documented.
Giansanti et. al. (2016) optimized protocols for six alternative proteases that have already shown promise in their applicability in proteomics, namely chymotrypsin, Lys-C, Lys-N, Asp-N, Glu-C and Arg-C have been created.
There are certain instances when trypsin does not provide adequate proteolysis. For example, many membrane proteins have limited number of tryptic cleavage sites. In other cases, distribution of tryptic cleavage sites is suboptimal, resulting in peptides that are too long or too short for mass spectrometry analysis. Promega offers various alternative proteases that complement trypsin and allow efficient protein analysis with mass spectrometry. Figure 5 highlights the benefits of the alternative protease chymotrypsin for protein mass spectrometry analysis.
Figure 5. Increased protein coverage using both trypsin and chymotrypsin.
Arg-C, Sequencing Grade (Cat.# V1881), also known as clostripain, is an endopeptidase that cleaves at the C-terminus of arginine residues, including sites next to proline. Arg-C activity is optimal at pH 7.6–7.9.
Asp-N, Sequencing Grade (Cat.# V1621), is an endoproteinase that hydrolyzes peptide bonds at the N-terminal side of aspartic acid residues. Asp-N activity is optimal at pH 4.0–9.0.
rAsp-N, Mass Spec Grade (Cat.# VA1160), is a recombinant protease that was cloned from Stenotrophomonas maltophilia and purified from E. coli.
Chymotrypsin, Sequencing Grade (Cat.# V1061), is a serine endoproteinase derived from bovine pancreas. The protease preferentially hydrolyzes at the carboxyl side of aromatic amino acids: tyrosine, phenylalanine and tryptophan. Chymotrypsin activity is optimal at pH 7.0–9.0.
Glu-C, Sequencing Grade (Cat.# V1651), is a serine protease that cleaves specifically at the C-terminal side of glutamic acid residues in the presence of ammonium bicarbonate or ammonium acetate. In phosphate buffers, cleavage also occurs at aspartic acid residues. Glu-C activity is optimal at pH 4.0–9.0.
rLys-C, Mass Spec Grade (Cat.# V1671), is recombinant endoproteinase Lys-C expressed in E. coli. The sequence origin of rLys-C is Protease IV from Pseudomonas aeruginosa. Similarly to native Lys-C, rLys-C cleaves at the C-terminus of lysine residues with exceptional specificity and retains proteolytic activity under denaturing conditions. rLys-C activity is optimal at pH 8.0–9.0.
Endoproteinase Lys-C, Mass Spec Grade (Cat.# VA1170), is a highly-purified serine protease that hydrolyzes specifically at the carboxyl side of lysines. Lys-C retains proteolytic activity under strong protein denaturing conditions.
Elastase (Cat.# V1891) is a serine protease that preferentially cleaves at the C-terminus of alanine, valine, serine, glycine, leucine or isoleucine residues. Elastase activity is optimal at pH 9.0.
Pepsin (Cat.# V1959) preferentially cleaves at the C-terminus of phenylalanine, leucine, tyrosine and tryptophan residues. Pepsin activity is optimal at pH 1.0–3.0.
Thermolysin (Cat.# V4001) is a thermostable metalloproteinase. Thermolysin preferentially cleaves at the N-terminus of the hydrophobic residues leucine, phenylalanine, valine, isoleucine, alanine and methionine. The optimal digestion temperature range is 65–85°C. Thermolysin activity is optimal at pH 5.0–8.5.