Pyridylethylation of cysteine residues in proteins

A procedure is described for using an inert polyvinylidene difluoride type membrane (e.g., ProBlott) as a matrix for on-membrane gas-phase pyridylethylation and as a desalting (cleanup) support following the solution phase pyridylethylation of cysteine residues in proteins. As a desalting support for 200 pmol of cardiotoxin, the membrane-derived sample gave a better recovery of phenylthiohydantoin (PTH)-amino acids during N-terminal sequencing compared to the sample obtained by rpHPLC. Using on-membrane gas-phase pyridylethylation, followed by replacement of glass-fiber filter disc and membrane washing/drying in vacuo, it was possible to eliminate the broad background peak that coeluted with PTH-Ala in the HPLC chromatogram completely. Using this modified procedure, there was no evidence of N-terminal alkylation of the amino-termini of proteins.