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@ARTICLE{Soh:873536,
      author       = {Soh, Wai Tuck and Demir, Fatih and Dall, Elfriede and
                      Perrar, Andreas and Dahms, Sven O. and Kuppusamy, Maithreyan
                      and Brandstetter, Hans and Huesgen, Pitter F.},
      title        = {{E}xte{ND}ing {P}roteome {C}overage with {L}egumain as a
                      {H}ighly {S}pecific {D}igestion {P}rotease},
      journal      = {Analytical chemistry},
      volume       = {92},
      number       = {4},
      issn         = {1520-6882},
      address      = {Columbus, Ohio},
      publisher    = {American Chemical Society},
      reportid     = {FZJ-2020-00804},
      pages        = {2961-2971},
      year         = {2020},
      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 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.},
      cin          = {ZEA-3},
      ddc          = {540},
      cid          = {I:(DE-Juel1)ZEA-3-20090406},
      pnm          = {582 - Plant Science (POF3-582) / 553 - Physical Basis of
                      Diseases (POF3-553) / ProPlantStress - Proteolytic
                      processing in plant stress signal transduction and responses
                      to abiotic stress and pathogen attack (639905)},
      pid          = {G:(DE-HGF)POF3-582 / G:(DE-HGF)POF3-553 /
                      G:(EU-Grant)639905},
      typ          = {PUB:(DE-HGF)16},
      pubmed       = {pmid:31951383},
      UT           = {WOS:000514758900014},
      doi          = {10.1021/acs.analchem.9b03604},
      url          = {https://juser.fz-juelich.de/record/873536},
}