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000873536 1001_ $$00000-0003-0082-7983$$aSoh, Wai Tuck$$b0
000873536 245__ $$aExteNDing Proteome Coverage with Legumain as a Highly Specific Digestion Protease
000873536 260__ $$aColumbus, Ohio$$bAmerican Chemical Society$$c2020
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000873536 520__ $$aBottom-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.
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000873536 7001_ $$0P:(DE-Juel1)176604$$aKuppusamy, Maithreyan$$b5$$ufzj
000873536 7001_ $$00000-0002-6089-3045$$aBrandstetter, Hans$$b6
000873536 7001_ $$0P:(DE-Juel1)162356$$aHuesgen, Pitter F.$$b7$$eCorresponding author
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