Publication Information
Soh et al., 2020
Abstract
Anal Chem. 2020 Feb 18;92(4):2961-2971. doi: 10.1021/acs.analchem.9b03604. Epub
2020 Jan 31.
ExteNDing Proteome Coverage with Legumain as a Highly Specific Digestion
Protease.
Soh WT(1), Demir F(2), Dall E(1), Perrar A(2), Dahms SO(1), Kuppusamy M(2),
Brandstetter H(1), Huesgen PF(2)(3)(4).
Author information:
(1)Department of Biosciences , University of Salzburg , 5020 Salzburg , Austria.
(2)Central Institute for Engineering, Electronics and Analytics, ZEA-3 ,
Forschungszentrum Jülich , 52428 Jülich , Germany.
(3)Cologne Excellence Cluster on Cellular Stress Responses in Aging Associated
Diseases, Medical Faculty and University Hospital , University of Cologne ,
50931 Cologne , Germany.
(4)Institute for Biochemistry, Faculty of Mathematics and Natural Sciences ,
University of Cologne , 50674 Cologne , Germany.
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.
DOI: 10.1021/acs.analchem.9b03604
PMCID: PMC7075662
PMID: 31951383 [Indexed for MEDLINE]
Conflict of interest statement: The authors declare no competing financial
interest.
