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首页> 外文期刊>Journal of mass spectrometry: JMS >Mass spectrometry of peptides and proteins using digestion by a grape cysteine protease at pH 3
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Mass spectrometry of peptides and proteins using digestion by a grape cysteine protease at pH 3

机译：在pH3下通过葡萄半胱氨酸蛋白酶消化的肽和蛋白质的质谱

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Cysteine protease from grapevine (Vitis vinifera) belongs to those resistant proteins, which survive the process of vinification and can therefore be detected as wine components. Its amino acid sequence shows a homology to other members of the papain family, but the enzyme has only partially been explored so far. In order to get more biochemical information with the help of mass spectrometry (MS), wine proteins were collected by ultrafiltration and separated by gel permeation chromatography. The purified enzyme surprisingly displayed a high molecular mass value of around 200 kDa, indicating a possible oligomeric status and aggregation, as it entered only negligibly the separating 10% gel during polyacrylamide gel electrophoresis. The isoelectric point (pI) value of 3.6 was determined by chromatofocusing. Matrix-assisted laser desorption/ionization (MALDI)-MS was employed to evaluate the cleavage specificity and usefulness of the isolated cysteine protease in protein and peptide research. A potential applicability could be anticipated from the efficient digestion performance in volatile ammonium formate buffers at pH 3. Common peptides were digested and the resulting products analyzed by MS/MS sequencing. Then, mixtures of protein standards and extracted barley nuclear proteins were processed in the same way. Grape cysteine protease is nonspecific but shows a certain preference for Arg, Lys, and also Leu residues. Compared with papain, it seems not to require fully the presence of a large hydrophobic residue adjacent to that at the cleavage site. The enzyme is suitable for protein research as it produces peptides of a reasonable length in acidic pH.
机译：从葡萄葡萄葡萄树（血管血管）属于那些抗性蛋白质的半胱氨酸蛋白酶，其在液化过程中存活，因此可以被检测为葡萄酒组分。它的氨基酸序列显示了木瓜蛋白酶家庭的其他成员的同源性，但到目前为止，酶仅部分探讨。为了通过质谱（MS）的帮助获得更多的生化信息，通过超滤收集葡萄酒蛋白并通过凝胶渗透色谱分离。纯化的酶令人惊讶地显示出大约200kDa的高分子质量值，表明可能的低聚状态和聚集，因为它在聚丙烯酰胺凝胶电泳期间仅忽略了分离的10％凝胶。通过染色体焦，确定3.6的等电点（PI）值。采用基质辅助激光解吸/电离（MALDI）-MS来评估蛋白质和肽研究中分离的半胱氨酸蛋白酶的切割特异性和有用性。可以从pH 3的挥发性铵甲酸盐缓冲液中的有效消化性能预期潜在的适用性。消化常见的肽和通过MS / MS测序分析的所得产物。然后，以相同的方式加工蛋白质标准和提取的大麦核蛋白的混合物。葡萄半胱氨酸蛋白酶是非特异性的，但表现出对Arg，Lys和Leu残基的某种偏好。与木瓜蛋白酶相比，似乎不需要完全存在与在切割位点相邻的大型疏水残留物。酶适用于蛋白质研究，因为它在酸性pH中产生合理长度的肽。

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