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.

Nucleosome scaffolding by Brd4 tandem bromodomains in acetylation-dependent chromatin compartmentalization

“…Binding to multiple mononucleosomes should increase their apparent size and therefore their sedimentation rate, shifting them down in a sucrose gradient (Figure S5). Consistent with the ability of Brd4 to access acetylation-dependent internucleosome interactions, addition of Brd4 (aa 36-460) to calf thymus mono-nucleosomes harboring high levels of lysine acetylation (57, 58) resulted in an increased sedimentation rate indicative of larger effective size similar to polynucleosomes relative to control without Brd4 addition (Figure 3A-B).…”

A comprehensive map of human elastin cross‐linking during elastogenesis

“…Purified proteases (nepenthesins and neprosin) [67] from the pitcher secretions of the tropical pitcher plant (Nepenthes sp.) Porcine pancreatic elastase (PE) and human cathepsin G (CG) were purchased from Elastin Products Company (Owensville, MO, USA).…”

“…Doubly distilled water (ddH 2 O) was used and all other chemicals were of analytical grade or higher purity. Purified proteases (nepenthesins and neprosin) [67] from the pitcher secretions of the tropical pitcher plant (Nepenthes sp.) [68] were a courtesy of D. Schriemer (University of Calgary, AB, Canada).…”

Unusual genome expansion and transcription suppression in ectomycorrhizal Tricholoma matsutake by repetitive insertions of transposable elements

“…They were mainly distributed over ascomycete genomes, and only a few basidiomycete genomes possessed them (S10–S12 Figs). Neprosin, a peptidase that cleaves C-terminal to proline residues under highly acidic conditions [27], was usually found in plant genomes, and few bacterial genomes have it. Only the Tricholoma and Laccaria genomes contained them among fungi.…”