title: Neprosin, a Selective Prolyl Endoprotease for Bottom-up Proteomics and Histone Mapping.
abstract: Trypsin dominates bottom-up proteomics, but there are reasons to consider alternative enzymes. Improving sequence coverage, exposing proteomic "dark matter," and clustering post-translational modifications in different ways and with higher-order drive the pursuit of reagents complementary to trypsin. Additionally, enzymes that are easy to use and generate larger peptides that capitalize upon newer fragmentation technologies should have a place in proteomics. We expressed and characterized recombinant neprosin, a novel prolyl endoprotease of the DUF239 family, which preferentially cleaves C-terminal to proline residues under highly acidic conditions. Cleavage also occurs C-terminal to alanine with some frequency, but with an intriguingly high "skipping rate." Digestion proceeds to a stable end point, resulting in an average peptide mass of 2521 units and a higher dependence upon electron-transfer dissociation for peptide-spectrum matches. In contrast to most proline-cleaving enzymes, neprosin effectively degrades proteins of any size. For 1251 HeLa cell proteins identified in common using trypsin, Lys-C, and neprosin, almost 50% of the neprosin sequence contribution is unique. The high average peptide mass coupled with cleavage at residues not usually modified provide new opportunities for profiling clusters of post-translational modifications. We show that neprosin is a useful reagent for reading epigenetic marks on histones. It generates peptide 1-38 of histone H3 and peptide 1-32 of histone H4 in a single digest, permitting the analysis of co-occurring post-translational modifications in these important N-terminal tails.
authors.affiliation: From the ‡Department of Biochemistry and Molecular Biology, University of Calgary, Calgary, Alberta T2N4N1, Canada.
authors.forename: Christoph U
authors.initials: CU
authors.lastname: Schräder
authors.affiliation: From the ‡Department of Biochemistry and Molecular Biology, University of Calgary, Calgary, Alberta T2N4N1, Canada.
authors.forename: Linda
authors.initials: L
authors.lastname: Lee
authors.affiliation: From the ‡Department of Biochemistry and Molecular Biology, University of Calgary, Calgary, Alberta T2N4N1, Canada.
authors.forename: Martial
authors.initials: M
authors.lastname: Rey
authors.affiliation: From the ‡Department of Biochemistry and Molecular Biology, University of Calgary, Calgary, Alberta T2N4N1, Canada.
authors.forename: Vladimir
authors.initials: V
authors.lastname: Sarpe
authors.affiliation: §BioCev-Institute of Microbiology, Czech Academy of Sciences, Vestec, Czech Republic 117 20.|¶Department of Biochemistry, Faculty of Science, Charles University in Prague, Prague, Czech Republic 116 36.
authors.forename: Petr
authors.initials: P
authors.lastname: Man
authors.affiliation: ‖Thermo Fisher Scientific, San Jose, California 95134.
authors.forename: Seema
authors.initials: S
authors.lastname: Sharma
authors.affiliation: ‖Thermo Fisher Scientific, San Jose, California 95134.
authors.forename: Vlad
authors.initials: V
authors.lastname: Zabrouskov
authors.affiliation: **Lunenfeld-Tanenbaum Research Institute, Sinai Health System, Toronto, Ontario, Canada M5G 1X5; and.
authors.forename: Brett
authors.initials: B
authors.lastname: Larsen
authors.affiliation: From the ‡Department of Biochemistry and Molecular Biology, University of Calgary, Calgary, Alberta T2N4N1, Canada; dschriem@ucalgary.ca.|‡‡Department of Chemistry, University of Calgary, Calgary, Alberta, Canada T2N 4N1.
authors.forename: David C
authors.initials: DC
authors.lastname: Schriemer
mesh_terms: D006367:HeLa Cells; D006657:Histones / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D010447:Peptide Hydrolases / Q000378:metabolism; D010455:Peptides / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D040901:Proteomics / Q000379:methods*; D011994:Recombinant Proteins / Q000378:metabolism
chemical_list: D006657:Histones; D010455:Peptides; D011994:Recombinant Proteins; D010447:Peptide Hydrolases