Modification of cysteine residues by alkylation. A tool in peptide mapping and protein identification
Modification of cysteine residues by alkylation. A tool in peptide mapping and protein identification
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DOI：
10.1021/ac9806005
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发表时间：
1998-12-15
影响因子：
7.4
通讯作者：
Chait, BT
中科院分区：
文献类型：
--
作者：
Sechi, S;Chait, BT
虽然质谱肽图谱已成为快速鉴定聚丙烯酰胺凝胶电泳（PAGE）分离的蛋白质的成熟技术，但鉴定程序的结果有时可能不明确。对于作为混合物分离的蛋白质或当仅分离非常少量的蛋白质时，这种模糊性变得越来越普遍。可以通过增加从凝胶中提取的肽的数量来提高鉴定程序的质量。在这里，我们表明需要半胱氨酸烷基化来确保通过PAGE分离的蛋白质的基质辅助激光解吸/电离飞行时间质谱（MALDI-TOF MS）肽图谱的最大覆盖范围。在所述程序中，在电泳前进行烷基化，以避免电泳期间丙烯酰胺加合物的偶然形成。以这种方式，用三种不同的烷基化试剂（4-乙烯基吡啶、碘乙酰胺、丙烯酰胺）获得均相烷基化。半胱氨酸烷基化也被用作鉴定含半胱氨酸肽的工具。使用未标记的丙烯酰胺和氘标记的丙烯酰胺（[2，3，3 '-D-3]丙烯酰胺）的1：1混合物，在电泳分离之前将感兴趣的蛋白质烷基化。通过MALDI-TOF MS分析由胰蛋白酶消化所得蛋白条带产生的肽混合物，并且从同位素分布推断肽的半胱氨酸含量。半胱氨酸含量信息很容易获得，并用于改善蛋白质鉴定过程。
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, acrylamide). Cysteine alkylation was also used as a tool for the identification of cysteine-containing peptides. Using a 1:1 mixture of unlabeled acrylamide and deuterium-labeled acrylamide ([2,3,3'-D-3]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 was inferred from the isotopic distributions. The cysteine content information was readily obtained and used to improve the protein identification process.