TY  - JOUR
AU  - Soh, Wai Tuck
AU  - Demir, Fatih
AU  - Dall, Elfriede
AU  - Perrar, Andreas
AU  - Dahms, Sven O.
AU  - Kuppusamy, Maithreyan
AU  - Brandstetter, Hans
AU  - Huesgen, Pitter F.
TI  - ExteNDing Proteome Coverage with Legumain as a Highly Specific Digestion Protease
JO  - Analytical chemistry
VL  - 92
IS  - 4
SN  - 1520-6882
CY  - Columbus, Ohio
PB  - American Chemical Society
M1  - FZJ-2020-00804
SP  - 2961-2971
PY  - 2020
AB  - 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.
LB  - PUB:(DE-HGF)16
C6  - pmid:31951383
UR  - <Go to ISI:>//WOS:000514758900014
DO  - DOI:10.1021/acs.analchem.9b03604
UR  - https://juser.fz-juelich.de/record/873536
ER  -