Universität zu Köln

Soh, Wai Tuck ORCID: 0000-0003-0082-7983, Demir, Fatih ORCID: 0000-0002-5744-0205, Dall, Elfriede ORCID: 0000-0002-0823-8983, Perrar, Andreas ORCID: 0000-0003-3365-5575, Dahms, Sven O., Kuppusamy, Maithreyan ORCID: 0000-0001-6866-0417, Brandstetter, Hans ORCID: 0000-0002-6089-3045 and Huesgen, Pitter F. (2020). ExteNDing Proteome Coverage with Legumain as a Highly Specific Digestion Protease. Anal. Chem., 92 (4). S. 2961 - 2972. WASHINGTON: AMER CHEMICAL SOC. ISSN 1520-6882

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Abstract

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 tlialiana 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.

Item Type:	Journal Article
Creators:	Creators Email ORCID ORCID Put Code Soh, Wai Tuck UNSPECIFIED orcid.org/0000-0003-0082-7983 UNSPECIFIED Demir, Fatih UNSPECIFIED orcid.org/0000-0002-5744-0205 UNSPECIFIED Dall, Elfriede UNSPECIFIED orcid.org/0000-0002-0823-8983 UNSPECIFIED Perrar, Andreas UNSPECIFIED orcid.org/0000-0003-3365-5575 UNSPECIFIED Dahms, Sven O. UNSPECIFIED UNSPECIFIED UNSPECIFIED Kuppusamy, Maithreyan UNSPECIFIED orcid.org/0000-0001-6866-0417 UNSPECIFIED Brandstetter, Hans UNSPECIFIED orcid.org/0000-0002-6089-3045 UNSPECIFIED Huesgen, Pitter F. UNSPECIFIED UNSPECIFIED UNSPECIFIED
URN:	urn:nbn:de:hbz:38-344588
DOI:	10.1021/acs.analchem.9b03604
Journal or Publication Title:	Anal. Chem.
Volume:	92
Number:	4
Page Range:	S. 2961 - 2972
Date:	2020
Publisher:	AMER CHEMICAL SOC
Place of Publication:	WASHINGTON
ISSN:	1520-6882
Language:	English
Faculty:	Unspecified
Divisions:	Unspecified
Subjects:	no entry
Uncontrolled Keywords:	Keywords Language MASS-SPECTROMETRY; COMPUTATIONAL PLATFORM; MYROSINASES TGG1; ACTIVATION; ENRICHMENT; DATABASE; PROGRESS; REVEALS; TERMINI; SITES Multiple languages Chemistry, Analytical Multiple languages
URI:	http://kups.ub.uni-koeln.de/id/eprint/34458