MOD OF CYSTEINE RESIDUES BY ALKYLATION TOOL IN PEPTIDE MAPPING & PROTEIN ID

肽图谱中烷基化工具对半胱氨酸残基的修饰

• 批准号: 6118326
• 负责人: Salvatore Sechi
• 金额: $ 0.85万
• 依托单位: ROCKEFELLER UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-12-10 至 1999-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6118326
• 关键词: biomedical resource; human tissue; nervous system; spectrometry

Although mass spectrometric peptide mapping has become an established technique for the rapid identification of proteins isolated by polyacrylamide gel electrophoresis (PAGE), the results of the identification procedure can sometimes be ambiguous. Such ambiguities become increasingly prevalent for proteins isolated as mixtures or when only very small amounts of the proteins are isolated. The quality of the identification procedure can be improved by increasing the number of peptides that are extracted from the gel. Here we show that cysteine alkylation is required to ensure maximal coverage in matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS) peptide mapping of proteins isolated by PAGE. In the described procedure, alkylation was performed prior to electrophoresis to avoid the adventitious formation of acrylamide adducts during electrophoresis. In this way, homogeneous alkylation was obtained with three different alkylating reagents (4 vinylpyridine, iodoacetamide, and acrylamide). Cysteine alkylation was also used as a tool for the identification of cysteine-containing peptides. Using a 11 mixture of unlabeled acrylamide and deuterium labeled acrylamide (2, 3, 3'-D3-acrylamide), the proteins of interest were alkylated prior to electrophoretic separation. Peptide mixtures produced by trypsin digestion of the resulting protein bands were analyzed by MALDI-TOF-MS and the cysteine content of the peptides were inferred from the isotopic distribution. The cysteine content information was readily obtained and used to improve the protein identification process. A paper describing these results is In press in Anal. Chem.

虽然质谱肽图谱已经成为一种 快速鉴定蛋白质的既定技术 通过聚丙烯酰胺凝胶电泳（PAGE）分离， 识别程序有时可能是模糊的。 等 对于分离的蛋白质， 混合物或当仅分离非常少量的蛋白质时。 鉴定程序的质量可以通过以下方式提高： 增加从凝胶中提取的肽的数量。 在这里，我们表明，半胱氨酸烷基化是必要的，以确保最大的 基质辅助激光解吸/电离飞行时间覆盖率 分离蛋白质的质谱（MALDI-TOF-MS）肽图谱 的PAGE。 在所描述的程序中，烷基化在以下步骤之前进行： 电泳以避免丙烯酰胺的偶然形成 电泳过程中的加合物。 这样，均相烷基化 用三种不同的烷基化试剂（4 乙烯基吡啶、碘乙酰胺和丙烯酰胺）。 半胱氨酸烷基化 还用作鉴定含半胱氨酸的工具 缩氨酸 使用11未标记的丙烯酰胺和氘的混合物 标记的丙烯酰胺（2，3，3 '-D3-丙烯酰胺），目的蛋白 在电泳分离之前烷基化。 肽混合物 通过胰蛋白酶消化得到的蛋白条带产生， 通过MALDI-TOF-MS分析肽的半胱氨酸含量， 从同位素分布推断。 半胱氨酸含量 信息很容易获得，并用于改善蛋白质 识别过程。 描述这些结果的一篇论文正在印刷中 在肛门。 Chem.