A strategy to locate cysteine residues in proteins by specific chemical cleavage followed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry

A simple methodology has been developed to characterize the number and location of free cysteine and cystine groups in peptides and proteins, using chemical modification and matrix-assisted laser desorption/ionization time-of flight mass spectrometry (MALDI-TOF MS), This new approach employs a specific reaction between free sulfhydryls and 2-nitro-5-thiocyanobenzoic acid (NTCB) to selectively cyanylate cysteine thiols, The N-terminal peptide bond of the modified cysteinyl residue can then be cleaved under alkaline conditions to form an amino-terminal peptide and a series of 2-iminothiazolidine-4-carboxylyl peptides which can be mapped to the sequence by MALDI-MS, The number and location of cysteines can be deduced from mass analysis of the peptide mixture resulting from NTCB chemical processing of the original protein of known sequence under nonreducing conditions, Similar experiments are then performed following disulfide bond reduction to further characterize both cysteine and cystine groups, Experimental conditions are described for protein disulfide bond reduction, sulfhydryl cyanylation, and cleavage reactions performed both in solution and on Zetabind membranes. (C) 1996 Academic Press, Inc.