Assessing alternative proteases for quantitative proteomics

Enzymatic proteolysis is a key step in mass spectrometry based, bottom up, quantitative proteomics. The go-to protease in bottom-up quantitative proteomics is trypsin due to its high efficiency and specificity. While it indeed provides good coverage of large portions of the proteome, a tryptic digestion underrepresents entire sets of proteins such as membrane and acidic proteins, as well as biologically important posttranslational modifications (PTM) that either interfere with tryptic digestion (such as methylation) or occur in stretches devoid of trypsin targets (i.e mucin domains).

Complementing trypsin, other proteases are also used in proteomics such as chymotrypsin, AspN, GluC and more. These may provide complementary digestion to improve the identification of proteins and domains less amenable to trypsin. However, their suitability for quantification has not been determined. While it is known that most enzymes are less specific and efficient than trypsin, it is unclear how these differences affect quantification accuracy in bottom up proteomics experiments.

Here we compare the quantitative performance between the gold standard – trypsin – and other enzymes: LysC, chymotrypsin, AspN, GluC, lysargiNase and thermolysin, to assess their suitability for quantitative proteomics. The parameters assessed were the accuracy of quantification, the proteome coverage gained and reproducibility.