Cyclic Peptide Protomer Detection in the Gas Phase: Impact on CCS Measurement and Fragmentation Patterns

With the recent improvements in ion mobility resolution, it is now possible to separate small protomeric tautomers, called protomers. In larger molecules above 1000 Da such as peptides, a few studies suggest that protomers do exist as well and may contribute to their gas-phase conformational heterogeneity. In this work, we observed a CCS distribution that can be explained by the presence of protomers of surfactin, a small lipopeptide with no basic site. Following preliminary density functional theoretical calculations, several protonation sites in the gas phase were energetically favorable in positive ionization mode. Experimentally, at least three near-resolved IM peaks were observed in positive ionization mode, while only one was detected in negative ionization mode. These results were in good agreement with the DFT predictions. CID breakdown curve analysis after IM separation showed different inflection points (CE₅₀) suggesting that different intramolecular interactions were implied in the stabilization of the structures of surfactin. The fragment ratio observed after collision-induced fragmentation was also different, suggesting different ring-opening localizations. All these observations support the presence of protomers on the cyclic peptide moieties of the surfactin. These data strongly suggest that protomeric tautomerism can still be observed on molecules above 1000 Da if the IM resolving power is sufficient. It also supports that the proton localization involves a change in the 3D structure that can affect the experimental CCS and the fragmentation channels of such peptides.