Modification of cysteine residues by alkylation. A tool in peptide mappingand protein identification

Although mass spectrometric peptide mapping has become an established techn ique for the rapid identification of proteins isolated by polyacrylamide ge l electrophoresis (PAGE), the results of the identification procedure can s ometimes be ambiguous. Such ambiguities become increasingly prevalent for p roteins isolated as mixtures or when only very small amounts of the protein s 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 spectromet ry (MALDI-TOF MS) peptide mapping of proteins isolated by PAGE. In the desc ribed procedure, alkylation was performed prior to electrophoresis to avoid the adventitious formation of acrylamide adducts during electrophoresis. I n this way, homogeneous alkylation was obtained with three different alkyla ting reagents (4-vinylpyridine, iodoacetamide, acrylamide). Cysteine alkyla tion 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 alkylat ed prior to electrophoretic separation. Peptide mixtures produced by trypsi n digestion of the resulting protein bands were analyzed by MALDI-TOF MS, a nd the cysteine content of the peptides was inferred from the isotopic dist ributions. The cysteine content information was readily obtained and used t o improve the protein identification process.