Successful de novo antibody sequencing depends on full coverage of the protein of interest that is best achieved through repeated identification of amino acid in different peptides with overlapping sequences; different proteases with different cleavage site rules can be used to make de novo antibody protein sequencing a success.
Less specific proteases such as chymotrypsin and pepsin generate more overlapping peptides than more specific proteases such as trypsin and Lys-C. This explains why studies have shown that employing a lower number of proteases can result in a higher amino acid coverage.
However, we observed that the presence of proline can result in inefficient cutting by trypsin, chymotrypsin, Asp-N, and pepsin. The recently commercially available A/P protease is capable of cutting peptides at C1 proline sites. Our findings show that the A/P protease can be used as a complementary tool to de novo protein sequencing. Particularly, in the case of antibody sequencing, additional proteases will be important for targeting conserved amino acid or specific motifs to facilitate their sequencing. We found that this was especially important for the CDR3 region, which is often a difficult-to-sequence antibody area.
This case study was adapted, with permission, from Le Bihan, T., Taylor, P., McDonald, Z., Liu, Q., Shen, J., Gorospe, K., Xu, X., Hosfield, C., Ma, B. (2019). Increased De Novo Protein Sequencing Coverage with Optimal Protease Cocktail. ASMS 2019 Atlanta, TP 020, with permission.