Authors: Andréa Mc Cann | Christopher Kune | Johann Far | Philippe Massonnet | Marc Ongena | Philippe Jacques | Gauthier Eppe | Loïc Quinton | Edwin De Pauw
Source: Open Repository and Bibliography (University of Liège) (article, 2019-06-01)
Field: Chemistry — Spectroscopy

Computational chemistry and ion mobility – mass spectrometry at high resolving power suggest prototropism of cyclic lipopeptides Introduction Cyclic lipopeptides (CLPs) are cyclic hydrophilic peptides with a lipid ramification using a β-hydroxy fatty acid that are produced by bacteria in a ribosome independent manner. Despite CLPs have relatively low molecular weight between 800 and 2,000 Da, the analysis of lipopeptides remains challenging due to the wide variety of synthetized isoforms differing in fatty acid chain length, in methyl group branching position, and in the nature of the amino-acids residues. These isoforms are suspected to have different biological activities requiring development of reliable methods for CLPs characterization. We present here an original approach combining UPLC and ion mobility - mass spectrometry at high resolving powers to separate the different species. Experimentally determined CCS will be compared with theoretical ones. Methods Lipopeptides were separated by UPLC (I-class, Waters, U.K.) on a C18 BEH column and identified by CID MS/MS mass spectrometry. Ion mobility – mass spectrometry (IM-MS) measurements were performed on a traveling wave ion mobility mass spectrometer (Synapt G2 HDMS from Waters, U.K.) and on a trapped Ion Mobility Mass Spectrometer (timsTOF, Bruker Daltonics, U.S.A.) to investigate the 3D structures of the ionized lipopeptides. Accurate Collison Cross Section (CCS) were obtained in both positive and negative mode and compared with theoretical CCS. Density Functional Theory (Gaussian) was used for structure optimizations at the CAM-B3LYP level of theory and 3-21G as basis set. Theoretical CCS have been computed from optimized structures using the trajectory method from IMoS V2. Preliminary data Separation of lipopeptides such as surfactins was successfully performed by reverse phase liquid chromatography. Lipopeptides were separated according to lipidic chain length and the branching position of the methyl group (iso/anteiso/linear). In positive ionization mode, the infusion of each isolated isoforms in the Synapt G2 showed a broad IMS distribution. These ion mobility profiles suggested the presence of different conformers. The higher IMS resolving power of the TIMS allowed the detection of at least three near-resolved peaks for a single isomer. In negative ionization mode however, only one peak was observed in the IM-MS profile on both Synapt G2 and TIMS, corresponding to one CCS. We included prototropic hypotheses where all the potential protonation and deprotonation sites on each lipopeptide had been determined by theoretical calculation. The abundances of the species in the CCS distributions of the resulting structures were obtained based on the Boltzmann distribution. Regarding the surfactin family, preliminary calculations by DFT shows that several protonation sites are energetically favorable and that the proton localization has a significant effect on the resulting CCS (∆CCS = 10Å²). These results are in good agreement with the experimental IMS profiles, obtained in both positive and negative ionization mode. Lipopeptides are then not related to a unique CCS value but a set of IM-MS profile that probably contains additional structural and physicochemical information. Novel aspect Experimental and theoretical approaches for lipopeptides IMS profiles analysis: protonation site determination, peaks intensity prediction and structural information extraction. Chemistry ; Mass spectrometry ; Ion-mobility spectrometry ; Ion-mobility spectrometry–mass spectrometry ; Ion ; Tandem mass spectrometry ; Chromatography ; Selected reaction monitoring ; Organic chemistry Mass Spectrometry Techniques and Applications ; Metabolomics and Mass Spectrometry Studies ; Analytical Chemistry and Chromatography