FIG. 3.

An example of disulfide bond identification as determined by chemical modification and LC-MS/MS analysis. MS analysis of the peptide GNSFPCDRPPTCY (covering residues 14 to 26) of SINV E3 under nonreduced conditions is shown in panels A and C, and that under reduced conditions is shown in panels B and D. Under nonreduced conditions, the m/z for the doubly charged GNSFPCDRPPTCY precursor peptide is 777.7. The same peptide under reduced conditions has a double-charge m/z of 835.6. Initial base peak chromatograms showed good separation for both the nonreduced and reduced samples. Furthermore, selected ion chromatograms showed a distinct peak corresponding to each precursor peptide (data not shown). Data-dependent tandem mass spectra were recorded by acquiring a precursor mass spectrum followed by two tandem mass spectra of the two most intense ions from the precursor scan. (A) Mass spectrum showing the intact precursor under nonreduced conditions with m/z 777.7. (B) Mass spectrum showing the intact precursor under reduced conditions with m/z 835.6. In both spectra the precursor is the main component, indicating that no other peptide fragments were coeluted. (C) The tandem mass spectrum of the m/z 777.7 precursor (nonreduced samples). (D) The tandem mass spectrum of the m/z 835.6 precursor (reduced samples). The sequence-specific fragment ions in both panels C and D are labeled y₅ to y₁₁, and all match the calculated m/z values, consistent with the disulfide bond being retained in these fragments (C) or with the absence of a disulfide bond and the two Cys residues being alkylated (D).