Characterization of cysteine residues and disulfide bonds in proteins by liquid chromatography/electrospray ionization tandem mass spectrometry

Cysteine residues and disulfide bonds are important for protein structure a nd function. We have developed a simple and sensitive method For determinin g the presence of free cysteine (Cys) residues and disulfide bonded Cys res idues in proteins (<100 pmol) by liquid chromatography/electrospray ionizat ion tandem mass spectrometry (LC/ESI-MS/MS) in combination with protein dat abase searching using the program Sequest. Free Cys residues in a protein w ere labeled with PEO-maleimide biotin immediately followed by denaturation with 8 M urea. Subsequently, the protein was digested with trypsin or chymo trypsin and the resulting products were analyzed by capillary LC/ESI-MS/MS for peptides containing modified Cys and/or disulfide bonded Cys residues. Although the RIS method for identifying disulfide bonds has been routinely employed, methods to prevent thiol-disulfide exchange have not been well do cumented. Our protocol was found to minimize the occurrence of the thiol-di sulfide exchange reaction. The method was validated using well-characterize d proteins such as aldolase, ovalbumin, and beta-lactoglobulin A. We also a pplied this method to characterize Cys residues and disulfide bonds of beta 1,4-galactosyltransferase (five Cys), and human blood group A and B glycos yltransferases (four Cys), Our results demonstrate that beta 1,4-galactosyl transferase contains one free Cys residue and two disulfide bonds, which is in contrast to work previously reported using chemical methods for the cha racterization of free Cys residues, but is consistent with recently publish ed results from x-ray crystallography. Tn contrast to the results obtained for beta 1,4-galactosyltransferase, none of the Cgs residues in A and B gly cosyltransferases were found to be involved in disulfide bonds. Copy-right (C) 2000 John Wiley & Sons, Ltd.