Studying Membrane Protein–Lipid Specificity through Direct Native Mass Spectrometric Analysis from Tunable Proteoliposomes

Abstract

Native mass spectrometry (nMS) has emerged as a key analytical tool to study the organizational states of proteins and their complexes with both endogenous and exogenous ligands. Specifically, for membrane proteins, it provides a key analytical dimension to determine the identity of bound lipids and to decipher their effects on the observed structural assembly. We recently developed an approach to study membrane proteins directly from intact and tunable lipid membranes where both the biophysical properties of the membrane and its lipid compositions can be customized. Extending this, we use our liposome-nMS platform to decipher the lipid specificity of membrane proteins through their multiorganelle trafficking pathways.To demonstrate this, we used VAMP2 and reconstituted it in the endoplasmic reticulum (ER), Golgi, synaptic vesicle (SV), and plasma membrane (PM) mimicking liposomes. By directly studying VAMP2 from these customized liposomes, we show how the same transmembrane protein can bind to different sets of lipids in different organellar-mimicking membranes. Considering that the cellular trafficking pathway of most eukaryotic integral membrane proteins involves residence in multiple organellar membranes, this study highlights how the lipid-specificity of the same integral membrane protein may change depending on the membrane context. Further, leveraging the capability of the platform to study membrane proteins from liposomes with curated biophysical properties, we show how we can disentangle chemical versus biophysical properties, of individual lipids in regulating membrane protein assembly.

A proteome-wide quantitative platform for nanoscale spatially resolved extraction of membrane proteins into native nanodiscs

“…Our collaborative efforts have already demonstrated the potential of MAPs in elucidating the functional states of endogenous MPs and their influence on downstream cellular signaling pathways 23,36 . Furthermore, our development of native mass spectrometry platforms enables the direct detection of membrane protein-lipid assemblies from the membrane itself 37,38 . The integration of these orthogonal technologies with MAP-based extraction platforms facilitating the rapid extraction of target proteins and their native interactome holds immense potential across all facets of membrane biology.…”

A Critical Evaluation of Detergent Exchange Methodologies for Membrane Protein Native Mass Spectrometry

“…Therefore, even trace amounts of the starting detergent have the potential to have significant structural effects on the MP of interest . Recent work has emphasized the need to move beyond detergent micelles to mimetics that are more similar to the cellular membrane, such as bicelles, , nanodiscs, , liposomes, , and lipid vesicles . These mimetic environments are better able to preserve physiologically relevant lipid and ligand binding, allowing them to produce higher fidelity nMS data when compared to detergent micelles in some cases .…”