Session: Parallel session 9 - Top-down and Structural analysis

XLMS of antimicrobial peptides under membrane-mimetic environment

Noelle POTIER¹, Emilie HIRSCHLER¹, Elise GLATTARD², Yannis FRANCOIS¹, Burkhard BECHINGER², Emmanuelle LEIZE¹

¹Laboratoire de Spectrométrie de Masse des Interactions et des Systèmes - Université de Strasbourg - UMR7140, Strasbourg, France
²Laboratoire de Biophysique des membranes et RMN - Université de Strasbourg - UMR7177, Strasbourg, France

Objective: This work focuses on the structural characterisation of 2 antimicrobial peptides in a membrane mimetic environment. Using a simple system, it illustrates the biases likely to be introduced insidiously during a crosslinking (XL) mass spectrometry (MS) experiment. PGLa and Magainin2 are 2 amphipathic peptides exhibiting antimicrobial properties on their own but they show also synergism when added in 1/1 cocktail. While synergistic mechanism of action is not yet well understood we proposed to use XLMS approach to help deciphering this behavior.

Methods: XL experiments were carried out either in a detergent- or lipidic- medium. N-Hydroxysuccunimide esters DSS, DSG and DST were used as crosslinkers. The XL reaction was followed by MALDI and cross-linked peptides were analysed before- or after- enzymatic digestion with trypsin, chymotrypsin and pepsin by nanoLC-MS/MS (Easy-nLC1000 - QExactive+, ThermoFisher).

Results: MALDI easily allowed to follow the XL reaction. It showed formation of intramolecular crosslinked monomers but also for the first time an unambiguous heterodimer. Interestingly such heterodimer is only observed under membrane mimetic environment when proper folding of the peptides occurred (verified by circular dichroism). Identification of crosslinked sites proved to be unexpectedly difficult showing complications at each step (especially non-classical behavior in nanoLC, resistance to trypsin digestion). After optimisation, two intermolecular sites could be proposed and their structural relevance is undergoing. To overcome this difficulty, assays using capillary zone electrophoresis in coupling with MS are underway.

Performing the XL in lipidic vesicles (SUV, LUV, MVL) appeared much more difficult in term of XL efficiency even at high crosslinker ratio, although the heterodimer was still the only multimeric detected species. This reminds us that each membrane-mimetic media is not totally equivalent and may induce different structural behavior.