ExteNDing proteome coverage with legumain as highly specific digestion protease

Wai Tuck Soh, Fatih Demir, Elfriede Dall, Sven Dahms, Hans Brandstetter, Pitter F Huesgen

Publikation: KonferenzbeitragPoster

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 en-zyme 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 human 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 and of mouse embryonic fibro-blast cell cultures and Arabidopsis thaliana leaves. Generating peptides highly complemen-tary in sequence, yet similar in their biophysical properties, legumain enabled complementary proteome and protein sequence coverage as compared to trypsin or GluC. Importantly, legumain further enabled the identification and enrichment of protein N-termini not accessible in GluC- or trypsin-digested sam-ples. Legumain cannot cleave after glycosylated Asn residues, which enabled 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
OriginalspracheEnglisch
PublikationsstatusVeröffentlicht - 18 Sep 2019
VeranstaltungAustrian Proteomics and Metabolomics Research Symposium 2019 - Paris-Lodron-University of Salzburg, Salzburg, Österreich
Dauer: 18 Sep 201920 Sep 2019

Konferenz

KonferenzAustrian Proteomics and Metabolomics Research Symposium 2019
LandÖsterreich
OrtSalzburg
Zeitraum18/09/1920/09/19

Systematik der Wissenschaftszweige 2012

  • 104 Chemie
  • 106 Biologie

Zitieren

Soh, W. T., Demir, F., Dall, E., Dahms, S., Brandstetter, H., & Huesgen, P. F. (2019). ExteNDing proteome coverage with legumain as highly specific digestion protease. Postersitzung präsentiert bei Austrian Proteomics and Metabolomics Research Symposium 2019, Salzburg, Österreich.