Julie S. K. Strømberga, Tor Haugb, Morten B. Strøma, Terkel Hansena,c, Terje Vasskoga aDepartment of Pharmacy, Faculty of Health Sciences, UiT the Arctic university of Norway, Breivika, N-9037 Tromsø, Norway bNorwegian College of Fishery Science, Faculty of Biosciences, Fisheries and Economics, UiT the Arctic university of Norway, Tromsø, Norway cDepartment of Biotechnology and Nanomedicine, SINTEF Industry, Trondheim, Norway
Disulfide-rich peptides constitute an important class of bioactive compounds with considerable potential as lead structures in drug discovery. The presence of multiple cysteine residues allows peptides to form intramolecular disulfide bridges, resulting in conformationally more stable three-dimensional structures compared to linear analogues1,2. However, a peptide sequence containing more than two cysteines can form several disulfide connectivities, giving rise to structural isomers with potentially different biological activities. Accurate determination of disulfide connectivity is therefore essential for structure activity relationship studies and for the development of disulfide-rich peptides as therapeutic candidates.
In this study, a liquid chromatography (LC)-based workflow was developed to characterize the disulfide connectivity of a model peptide containing four cysteine residues. The peptide, an in-house synthesized truncated analogue of a bioactive marine peptide Turgencin A originating from Synoicum turgens, was found to form three structural isomers when oxidized, with distinct antimicrobial activities. The elucidation of disulfide connectivity integrates solid-phase disulfide reduction and stepwise alkylation with LC separation and tandem mass spectrometric (MS/MS) analysis. LC-MS/MS played a central role both in the development of the peptides, and elucidation based on their respective disulfide connectivities by identifying differential alkylation patterns with MS/MS fragmentation profiles. This study presents an optimized approach for disulfide connectivity elucidation, building on an established method by Albert et al.3
Minimum inhibitory concentration (MIC) assays against a panel of Gram-positive and Gram-negative bacteria revealed marked differences in antimicrobial potency between the LC-separated isomers, underscoring the biological significance of disulfide connectivity.
Overall, this work highlights the utility of LC, in combination with controlled chemical modification and mass spectrometric analysis, as a powerful analytical platform for elucidating disulfide connectivities in cysteine-rich peptides. The developed LC-based method provides a straightforward and robust approach for structural characterization that can be broadly applied in peptide drug discovery and natural product research.
References
1. Barry, D. G., Daly, N. L., Clark, R. J., Sando, L. & Craik, D. J. Linearization of a Naturally Occurring Circular Protein Maintains Structure but Eliminates Hemolytic Activity †,‡. 1, 6688–6695 (2003).
2. Aman, J. W. et al. Insights into the origins of fish hunting in venomous cone snails from studies of Conus tessulatus. Proc. Natl. Acad. Sci. U. S. A. 112, 5087–5092 (2015).
3. Albert, A. et al. General Approach to Determine Disulfide Connectivity in Cysteine-Rich Peptides by Sequential Alkylation on Solid Phase and Mass Spectrometry. Anal. Chem. 88, 9539–9546 (2016).