The utility of proteases in proteomics, from sequence profiling to structure and function analysis

Abstract In mass spectrometry (MS)‐based bottom‐up proteomics, protease digestion plays an essential role in profiling both proteome sequences and post‐translational modifications (PTMs). Trypsin is the gold standard in digesting intact proteins into small‐size peptides, which are more suitable for high‐performance liquid chromatography (HPLC) separation and tandem MS (MS/MS) characterization. However, protein sequences lacking Lys and Arg cannot be cleaved by trypsin and may be missed in conventional proteomic analysis. Proteases with cleavage sites complementary to trypsin are widely applied in proteomic analysis to greatly improve the coverage of proteome sequences and PTM sites. In this review, we survey the common and newly emerging proteases used in proteomics analysis mainly in the last 5 years, focusing on their unique cleavage features and specific proteomics applications such as missing protein characterization, new PTM discovery, and de novo sequencing. In addition, we summarize the applications of proteases in structural proteomics and protein function analysis in recent years. Finally, we discuss the future development directions of new proteases and applications in proteomics.