ExteNDing Proteome Coverage with Legumain as a Highly Specific Digestion Protease

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.