The characterization of peptides bearing multiple disulfide bridges requires sequence and a correct assignment of cysteine pairings. One major goal of the European Project Venomics is to develop a reliable high throughput method to fully characterize peptides in complex mixtures such as raw venoms. A specific emphasize is given to the determination of cysteine pairings. Ion mobility may bring an important contribution. It separates mass selected conformers on the basis of differences in drift time in the presence of collision gas. Those differences result from differences in collision cross sections that can be represented by the rotationally averaged projection of the ionic volume on a plane. Each “shape separated” peptide is then activated by CID and sequenced using MS/MS.
We modified the method to assign cysteine pairing inducing differences in cross section using eletron transfer reduction.This step consists in statistically opening only one disulfide bond1 . As verified by molecular modeling, once a disulfide bridge is opened, the structure rapidly expands. CID MS/MS of the opened moiety becomes more efficient. In the case of peptides bearing two disulfide bridges, two arrival time distributions can be observed for the singly reduced species, each corresponding to a specific connectivity. On the contrary to chemical reduction in solution, disulfide bond scrambling is less likely to occur. Differences in MS/MS spectra allow assigning the S-S pairing.
Applied to crude venoms, this LC/MS-ETD-IMS-CID-MS is a promising new fast online full characterization strategy, including SS pairing assignment. Figures of merit and limitations will be commented on the basis of examples involving species of increasing mass ranging from small toxins (<4kDa) in Conus snails type venoms to larger peptides (>4kDa) from snake venoms.