ExteNDing Proteome Coverage with Legumain as a Highly Specific Digestion Protease

Anal Chem. 2020 Feb 18;92(4):2961-2971. doi: 10.1021/acs.analchem.9b03604. Epub 2020 Jan 31.


Bottom-up mass spectrometry-based proteomics utilizes proteolytic enzymes with well characterized specificities to generate peptides amenable for identification by high-throughput tandem mass spectrometry. Trypsin, which cuts specifically after the basic residues lysine and arginine, is the predominant enzyme used for proteome digestion, although proteases with alternative specificities are required to detect sequences that are not accessible after tryptic digest. Here, we show that the human cysteine protease legumain exhibits a strict substrate specificity for cleavage after asparagine and aspartic acid residues during in-solution digestions of proteomes extracted from Escherichia coli, mouse embryonic fibroblast cell cultures, and Arabidopsis thaliana leaves. Generating peptides highly complementary in sequence, yet similar in their biophysical properties, legumain (as compared to trypsin or GluC) enabled complementary proteome and protein sequence coverage. Importantly, legumain further enabled the identification and enrichment of protein N-termini not accessible in GluC- or trypsin-digested samples. Legumain cannot cleave after glycosylated Asn residues, which enabled the robust identification and orthogonal validation of N-glycosylation sites based on alternating sequential sample treatments with legumain and PNGaseF and vice versa. Taken together, we demonstrate that legumain is a practical, efficient protease for extending the proteome and sequence coverage achieved with trypsin, with unique possibilities for the characterization of post-translational modification sites.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Arabidopsis / metabolism
  • Cysteine Endopeptidases / chemistry
  • Cysteine Endopeptidases / isolation & purification
  • Cysteine Endopeptidases / metabolism*
  • Escherichia coli / metabolism
  • Humans
  • Mice
  • Plant Leaves / metabolism
  • Proteome / chemistry
  • Proteome / metabolism*
  • Proteomics


  • Proteome
  • Cysteine Endopeptidases
  • asparaginylendopeptidase