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.
Showing the most recent 10 out of 67 publications
