Characterization of Protein Disulfide Linkages by MS In-Source Dissociation Comparing to CID and ETD Tandem MS
Direct characterization of disulfide linkages in proteins by mass spectrometry has been challenging. Here, we report analysis of disulfide linkages in insulin variant, endothelin 3, and relaxin 2 by in-source dissociation (ISD) during LC-MS. A duplet insulin peptide from Glu-C digestion that contains peptides p1 and p2 (from chains A and B, respectively) was selected as a model peptide. This duplet peptide has an inter-chain disulfide bond between p1 and p2, and an intra-chain disulfide bond in p1. To compare the gas-phase fragmentation, it was subjected to ISD MS and MS/MS methods, including collision-induced dissociation (CID) and electron transfer dissociation (ETD). The pattern and efficiency of peptide backbone and disulfide cleavage varied with these dissociation methods. ETD, CID, and ISD were able to generate single backbone, double backbone, and triple (double backbone and single disulfide bond) cleavages in this model peptide, respectively. Specifically, CID did not cleave disulfide bonds and ETD was able to only cleave the inter-chain disulfide bond at low efficiency, limiting their usage in this disulfide analysis. In contrast, ISD was able to cleave the intra-chain disulfide bond in addition to peptide backbone, creating multiple fragment ions that allow accurate assignment of both intra- and inter-chain disulfide linkages. ISD was also successfully applied to determine double disulfide linkages in endothelin 3 and relaxin 2 peptides. This study contributes to the fundamental understanding of disulfide bond cleavages in different gas-phase fragmentations and provides an efficient cleavage strategy for identification of disulfide bonds in proteins by ISD ESI-MS.
KeywordsDisulfide bond Insulin Relaxin 2 Endothelin 3 In-source dissociation (ISD) LC-MS CID ETD MS/MS Glu-C digestion Trypsin and Lys-C digestion
The authors thank Michael J. Iammarino for providing the sample.
- 1.Chang, S.G., Choi, K.D., Jang, S.H., Shin, H.C.: Role of disulfide bonds in the structure and activity of human insulin. Mol. Cells. 16, 323–330 (2003)Google Scholar
- 8.Wilkinson, T.N., Speed, T.P., Tregear, G.W., Bathgate, R.A.: Evolution of the relaxin-like peptide family. BMC Evol. Biol. 5, (2005)Google Scholar
- 32.Chelius, D., Wimer, M.E.H., Bondareriko, P.V.: Reversed-phase liquid chromatography in-line with negative ionization electrospray mass spectrometry for the characterization of the disulfide-linkages of an immunoglobulin gamma antibody. J. Am. Soc. Mass Spectrom. 17, 1590–1598 (2006)CrossRefGoogle Scholar
- 33.Zubarev, R.A., Kruger, N.A., Fridriksson, E.K., Lewis, M.A., Horn, D.M., Carpenter, B.K., McLafferty, F.W.: Electron capture dissociation of gaseous multiply-charged proteins is favored at disulfide bonds and other sites of high hydrogen atom affinity. J. Am. Chem. Soc. 121, 2857–2862 (1999)CrossRefGoogle Scholar