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Bottom-up mass spectrometry-based proteomics utilizes proteolytic enzymes with well character-ized 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 specificity are required to detect sequences that are not accessible after tryptic digest. Here, we show that the hu-man cysteine protease legumain exhibits strict substrate specificity for cleavage after asparagine and aspartic acid residues during in-solution digestions of proteomes extracted from E.coli, mouse em-bryonic fibroblast cell cultures and Arabidopsis thaliana leaves. Generating peptides highly com-plementary in sequence, yet similar in their biophysical properties, legumain enabled complemen-tary proteome and protein sequence coverage as compared to trypsin or GluC. Importantly, legu-main 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 ena-bled robust identification and orthogonal validation of N-glycosylation sites based on alternating sequential sample treatment 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.
This article was published in the following journal.
Name: Analytical chemistry
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Legumain (asparaginyl endopeptidase) is the only protease with a preference for cleavage after asparagine residues. Increased legumain activity is a hallmark of inflammation, neurodegenerative disease...
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Health insurance coverage for all persons in a state or country, rather than for some subset of the population. It may extend to the unemployed as well as to the employed; to aliens as well as to citizens; for pre-existing conditions as well as for current illnesses; for mental as well as for physical conditions.
Insurance coverage of a physical or mental health condition, disability, or illness existing before health insurance is applied for or enrolled in.
Insurance providing coverage of medical, surgical, or hospital care in general or for which there is no specific heading.
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