Schräder Christoph U, Lee Linda, Rey Martial, Sarpe Vladimir, Man Petr, Sharma Seema, Zabrouskov Vlad, Larsen Brett, Schriemer David C
From the ‡Department of Biochemistry and Molecular Biology, University of Calgary, Calgary, Alberta T2N4N1, Canada.
§BioCev-Institute of Microbiology, Czech Academy of Sciences, Vestec, Czech Republic 117 20.
Mol Cell Proteomics. 2017 Jun;16(6):1162-1171. doi: 10.1074/mcp.M116.066803. Epub 2017 Apr 12.
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.
胰蛋白酶在自下而上的蛋白质组学中占据主导地位，但仍有理由考虑使用其他酶。提高序列覆盖率、揭示蛋白质组学中的“暗物质”，以及以不同方式和更高层次对翻译后修饰进行聚类，推动了对与胰蛋白酶互补的试剂的探索。此外，易于使用且能产生更大肽段以利用更新的碎裂技术的酶，在蛋白质组学中也应占有一席之地。我们表达并表征了重组内蛋白酶，它是DUF239家族的一种新型脯氨酰内切蛋白酶，在高酸性条件下优先在脯氨酸残基的C端进行切割。在丙氨酸的C端也会有一定频率的切割，但具有极高的“跳跃率”，令人着迷。消化过程会达到一个稳定的终点，平均肽质量为2521个单位，且在肽谱匹配中对电子转移解离的依赖性更高。与大多数脯氨酸切割酶不同，内蛋白酶能有效降解任何大小的蛋白质。对于使用胰蛋白酶、Lys-C和内蛋白酶共同鉴定出的1251种HeLa细胞蛋白质，内蛋白酶贡献的序列中近50%是独特的。高平均肽质量以及在通常未修饰的残基处的切割，为翻译后修饰簇的分析提供了新机会。我们表明，内蛋白酶是用于读取组蛋白上表观遗传标记的有用试剂。它在一次消化中就能产生组蛋白H3的肽段1 - 38和组蛋白H4的肽段1 - 32，从而能够分析这些重要N端尾巴中共存的翻译后修饰。
