Cross-Linking Mass Spectrometry of the Antimicrobial Peptides Magainin 2 and PGLa Reveals Heterodimerization in Micellar Medium.

Rationale: In this study, we applied cross-linking mass spectrometry (XL-MS) to characterize the oligomeric states of a PGLa/magainin 2 mixture and gain insight into the heterodimerization previously suggested in the literature. Both peptides have shown a synergistic enhancement of activity when tested in antimicrobial assays; however, the mechanism of action is still not well understood.

Methods: Peptides solutions were prepared in HEPES buffer in the presence of membrane-mimicking DDM detergent micelles or POPE:POPG 3:1 vesicles. Cross-linking experiments were performed using disuccinimidyl suberate (DSS) or disuccinimidyl glutarate (DSG), and MALDI-MS was used to follow the cross-linking performance. Nano liquid chromatography coupled to mass spectrometry was conducted on a Q Exactive Plus orbitrap to achieve linkage sites determination using pLink2 for data interpretation. Trypsin or pepsin digestion was performed for the characterization of intermolecular links.

Results: XL-MS performed in a DDM micelle environment provided direct evidence of a specific PGLa/magainin 2 heterodimer, but no other oligomeric states were detected. Monitoring the reaction using MALDI-MS allowed unambiguous characterization of the cross-linked stabilized oligomers and facilitated a rapid optimization of conditions to achieve the best balance between stabilizing complex formation and avoiding unspecific aggregation. Comparison of the cross-linked species in detergent micelles and lipidic POPE:POPG bilayers revealed different behaviors suggesting that interaction between the peptides might occur differently in both membrane-mimicking media.

Conclusions: This study revealed that XL-MS was relevant at the peptidomic level. However, the cross-linking workflow had to be adjusted compared to its use in large-scale protein-protein interaction mapping in order to avoid technical bias arising from the rapid nature of the cross-linking reaction.