Ion Mobility–Mass Spectrometry of Lasso Peptides: Signature of a Rotaxane Topology

Abstract

Ion mobility mass spectrometry data were collected on a set of five class II lasso peptides and their branched-cyclic topoisomers prepared in denaturing solvent conditions with and without sulfolane as a supercharging agent. Sulfolane was shown not to affect ion mobility results and to allow the formation of highly charged multiply protonated molecules. Drift time values of low charged multiply protonated molecules were found to be similar for the two peptide topologies, indicating the branched-cyclic peptide to be folded in the gas phase into a conformation as compact as the lasso peptide. Conversely, high charge states enabled a discrimination between lasso and branched-cyclic topoisomers, as the former remained compact in the gas phase while the branched-cyclic topoisomer unfolded. Comparison of the ion mobility mass spectrometry data of the lasso and branched-cyclic peptides for all charge states, including the higher charge states obtained with sulfolane, yielded three trends that allowed differentiation of the lasso form from the branched-cyclic topology: low intensity of highly charged protonated molecules, even with the supercharging agent, low change in collision cross sections with increasing charge state of all multiply protonated molecules, and narrow ion mobility peak widths associated with the coexistence of fewer conformations and possible conformational changes.

Insights into the Unique Phosphorylation of the Lasso Peptide Paeninodin

“…S1). In agreement with a previous study (33), these results are explained by thermally induced unthreading, which yields a branched-cyclic peptide with a larger cross-section and therefore a longer drift time compared with the more compact lasso peptide. This analysis also yielded a pair of peaks, indicating that the peptide can adopt multiple, distinct, and interchangeable conformations in its native state.…”

“…Before analysis, the samples were dissolved in 50% MeCN containing an additional 4% formic acid and 0.5% sulfolane as supercharging agent. The measurements were performed with previously described instrument settings (33).…”

Ion Mobility Collision Cross Section Compendium

“…Also not reflected in the analysis presented in Figure 7 are the recent interests in characterizing natural products by IM-MS based CCS measurements. 69,116,238–240 Many natural products contain complex and unusual scaffolds which motivates their study by a structurally-selective technique such as IM-MS, however, natural products are conventionally classified based on bioactivity rather than structure and as such molecules which can be considered natural products are represented in virtually all of the chemical classes delineated in this review. A similar issue is seen in metabolites (not shown) which is a classification that includes small peptides, carbohydrates and lipids.…”

Thermal Unthreading of the Lasso Peptides Astexin-2 and Astexin-3

“…26 It has also been demonstrated that ion mobility mass spectrometry can differentiate between the threaded and unthreaded states of lasso peptides in high charge states. 27 Despite this large body of work, there is still no detailed analysis of the energetics and kinetics of lasso peptide unthreading.…”