Native mass spectrometry of membrane proteins reconstituted in peptidiscs

Abstract

Membrane proteins and lipids are essential for a wide range of cellular processes, making their structural characterisation essential for understanding biological function. However, the amphipathic nature of membrane proteins poses a significant challenge for traditional structural biology techniques. Membrane mimetics offer an alternative approach to studying membrane proteins in more native-like environments. Among them, peptidiscs have emerged as a promising tool for stabilising membrane proteins, allowing reconstitution from detergent micelles into a detergent-free, native-like environment that preserves structural integrity. While peptidiscs have shown utility in techniques such as mass photometry and cryo-EM, their compatibility with native mass spectrometry (MS) remains largely unexplored. In this study, we evaluate the feasibility of using peptidiscs for native MS analysis of membrane proteins and their complexes, focusing on the antibiotic resistance efflux pump AceI and the β-barrel assembly machinery (BAM complex). We reconstituted these proteins into peptidiscs using both ‘on-column’ and ‘on-bead’ assembly methods and assessed complex integrity and stability post-reconstitution using native MS. Our findings highlight the potential of peptidiscs as a tool for native MS-based structural characterisation of membrane protein and their assemblies.