Figure 2: Multiple protease proteolysis improves protein inference.

The use of other proteases beyond Trypsin such as Lysyl endopeptidase (Lys-C), Peptidyl-Asp metallopeptidase (Asp-N), Glutamyl peptidase I, (Glu-C), Chymotrypsin, Clostripain (Arg-C) or Peptidyl-Lys metalloendopeptidase (Lys-N) can generate a greater diversity of peptides. This improves protein sequence coverage and allows for the correct identification of their N-termini. Increasing the number of complimentary enzymes used will increase the number of proteins identified by single peptides and decreases the ambiguity of the assignment of protein groups. Therefore, this will allow more protein isoforms and post-translational modifications to be identified than using Trypsin alone.