In-gel s-pyridylethylation of gel-resolved proteins: individual spot method

INTRODUCTIONThis protocol describes a method for performing reduction and S-alkylation of Coomassie-blue-stained proteins within individually excised, stained protein spots or bands. Reduction is performed with dithiothreitol, and alkylation with 4-vinylpyridine. (Treatment of free cysteines with 4-vinylpyridine yields the S-β-(4-pyridylethyl) cysteinyl derivative.) S-β-(4-pyridylethyl) cysteine-containing peptides can be readily identified during RP-HPLC by their characteristic absorbance at 254 nm and during electrospray ionization tandem mass spectrometry by the appearance of a characteristic pyridylethyl fragment ion of 10(6) Da. The position of cysteine residues in a polypeptide sequence can be determined either by mass spectrometry or as phenylthiohydantoin S-β-(4-pyridylethyl) cysteine during Edman degradation.