Modification of Cysteine Residues by Alkylation. A Tool in Peptide Mapping and Protein Identification
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- 作者：Salvatore Sechi and Brian T. Chait
- 刊名：Analytical Chemistry
- 出版年：1998
- 出版时间：December 15, 1998
- 年：1998
- 卷：70
- 期：24
- 页码：5150 - 5158
- 全文大小：108K
- 年卷期：v.70,no.24(December 15, 1998)
- ISSN：1520-6882
文摘
Although mass spectrometric peptide mapping has become an established technique for the rapid identificationof proteins isolated by polyacrylamide gel electrophoresis(PAGE), the results of the identification procedure cansometimes be ambiguous. Such ambiguities becomeincreasingly prevalent for proteins isolated as mixturesor when only very small amounts of the proteins areisolated. The quality of the identification procedure canbe improved by increasing the number of peptides thatare extracted from the gel. Here we show that cysteinealkylation is required to ensure maximal coverage inmatrix-assisted laser desorption/ionization time-of-flightmass spectrometry (MALDI-TOF MS) peptide mapping ofproteins isolated by PAGE. In the described procedure,alkylation was performed prior to electrophoresis to avoidthe adventitious formation of acrylamide adducts duringelectrophoresis. In this way, homogeneous alkylation wasobtained with three different alkylating reagents (4-vinylpyridine, iodoacetamide, acrylamide). Cysteine alkylation was also used as a tool for the identification ofcysteine-containing peptides. Using a 1:1 mixture ofunlabeled acrylamide and deuterium-labeled acrylamide([2,3,3'-D3]acrylamide), the proteins of interest werealkylated prior to electrophoretic separation. Peptidemixtures produced by trypsin digestion of the resultingprotein bands were analyzed by MALDI-TOF MS, and thecysteine content of the peptides was inferred from theisotopic distributions. The cysteine content informationwas readily obtained and used to improve the proteinidentification process.