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

Abstract

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.

α-Lactalbumin (LA) Stimulates Milk β-1,4-Galactosyltransferase I (β4Gal-T1) to Transfer Glucose from UDP-glucose to N-Acetylglucosamine

“…In the crystal structure of wild-type Gal-T1, Cys-342 residue exists in the reduced form, whereas the remaining four Cys residues form two disulfide bonds. Recent mass spectroscopic studies have shown that, during denaturation of milk Gal-T1, this free Cys-342 residue is responsible for the formation of mixed disulfide bonds (29). Therefore, the increased in vitro folding efficiency of the mutant C342T-Gal-T1 may be attributed to the diminished mixed disulfide bond formation during folding.…”

Unique Disulfide Bond Structures Found in ST8Sia IV Polysialyltransferase Are Required for Its Activity

“…In the present study, we also determined the disulfide bond structures of ST8Sia IV using improved LC/ESI-MS/MS analysis (37,38). Our results demonstrated that the cysteine residue at the COOH-terminal end forms a disulfide bond with the second cysteine residue of sialylmotif L and that the second disulfide bond is formed between the first cysteine at sialylmotif L and the cysteine at sialylmotif S. These disulfide bonds are intramolecularly formed, because almost no homodimer was detected (Fig.…”

“…The resulting MS/MS spectra were then searched against a protein data base (Owl) by Sequest to confirm the sequence of tryptic or chymotryptic peptides. The details of the LC/ESI-MS/MS procedure were described elsewhere (38).…”

Mass spectrometry-based strategies for protein disulfide bond identification

“…However, the three-dimensional structure of proteins may hinder the access of NEM and decrease the blocking efficiency (Lind et al 2002). Thereby, denaturing reagents such as urea and guanidine hydrochloride are sometimes added to help unfold proteins (Yen et al 2000). The disadvantage of urea is it contains slight amount of cyanate, which reacts with thiol groups to form thiocarbamates (Stark et al 1960).…”