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 and function. We have developed a simple and sensitive method for determining the presence of free cysteine (Cys) residues and disulfide bonded Cys residues in proteins (<100 pmol) by liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) in combination with protein database searching using the program Sequest. Free Cys residues in a protein were labeled with PEO-maleimide biotin immediately followed by denaturation with 8 M urea. Subsequently, the protein was digested with trypsin or chymotrypsin 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 MS method for identifying disulfide bonds has been routinely employed, methods to prevent thiol-disulfide exchange have not been well documented. Our protocol was found to minimize the occurrence of the thiol-disulfide exchange reaction. The method was validated using well-characterized proteins such as aldolase, ovalbumin, and beta-lactoglobulin A. We also applied this method to characterize Cys residues and disulfide bonds of beta 1,4-galactosyltransferase (five Cys), and human blood group A and B glycosyltransferases (four Cys). Our results demonstrate that beta 1,4-galactosyltransferase contains one free Cys residue and two disulfide bonds, which is in contrast to work previously reported using chemical methods for the characterization of free Cys residues, but is consistent with recently published results from x-ray crystallography. In contrast to the results obtained for beta 1,4-galactosyltransferase, none of the Cys residues in A and B glycosyltransferases were found to be involved in disulfide bonds.
Yen
T Y
TY
Department of Chemistry and Biochemistry, San Francisco State University, San Francisco, California 94132, USA.
Joshi
R K
RK
Yan
H
H
Seto
N O
NO
Palcic
M M
MM
Macher
B A
BA
D000595:Amino Acid Sequence; D000818:Animals; D002853:Chromatography, Liquid / Q000379:methods*; D003545:Cysteine / Q000737:chemistry*; D004220:Disulfides / Q000737:chemistry*; D016695:Glycosyltransferases / Q000737:chemistry; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007782:Lactoglobulins / Q000737:chemistry; D013058:Mass Spectrometry / Q000379:methods*; D009237:N-Acetyllactosamine Synthase / Q000737:chemistry; D010047:Ovalbumin / Q000737:chemistry; D011489:Protein Denaturation; D011506:Proteins / Q000737:chemistry*
D004220:Disulfides; D007782:Lactoglobulins; D011506:Proteins; D010047:Ovalbumin; D016695:Glycosyltransferases; D009237:N-Acetyllactosamine Synthase; D003545:Cysteine