Identifying key membrane protein lipid interactions using mass spectrometry

Abstract

With the recent success in determining membrane protein structures, further detailed understanding of the identity and function of the bound lipidome is essential. Using an approach that combines high-energy native mass spectrometry (HE-nMS) and solution-phase lipid profiling, this protocol can be used to determine the identity of the endogenous lipids that directly interact with a protein. Furthermore, this method can identify systems in which such lipid binding has a major role in regulating the oligomeric assembly of membrane proteins. The protocol begins with recording of the native mass spectrum of the protein of interest, under successive delipidation conditions, to determine whether delipidation leads to disruption of the oligomeric state. Subsequently, we propose using a bipronged strategy: first, an HE-nMS platform is used that allows dissociation of the detergent micelle at the front end of the instrument. This allows for isolation of the protein-lipid complex at the quadrupole and successive fragmentation at the collision cell, which leads to identification of the bound lipid masses. Next, simultaneous coupling of this with in-solution LC-MS/MS-based identification of extracted lipids reveals the complete identity of the interacting lipidome that copurifies with the proteins. Assimilation of the results of these two sets of experiments divulges the complete identity of the set of lipids that directly interact with the membrane protein of interest, and can further delineate its role in maintaining the oligomeric state of the protein. The entire procedure takes 2 d to complete.

Modular detergents tailor the purification and structural analysis of membrane proteins including G-protein coupled receptors

“…In practice, the investigation of protein-lipid interaction by MS is addressed mainly in two ways: First, membrane proteins are delipidated step-wise with detergents that exhibit weak delipidating properties, such as DDM. To do so, protein-lipid complexes are repetitively purified by SEC, IMAC, or dialysis until mass spectra of sufficient quality are obtained 36,39 . This allows us to investigate membrane proteins in complex with co-purified membrane lipids 40 .…”

The energetics of protein–lipid interactions as viewed by molecular simulations

“…It is also evident that we need both more and better experimental data against which to evaluate and compare predictions from simulations. Data are needed both from in vitro experiments (ideally in model membranes rather than in detergents) [102][103][104] and from in vivo studies, e.g. single-molecule experiments in live cell membranes [105].…”

Cholesterol interaction sites on the transmembrane domain of the hedgehog signal transducer and Class F G protein-coupled receptor Smoothened

“…Such functional assays could either be conducted using signaling assays in cells , or indeed in minimal in vitro reconstituted nanodisc systems, where the activity of the truncated 7TMD has been shown to depend on cholesterol (Myers et al, 2017). Secondly, native mass spectrometry has shown recent tremendous utility in probing the specific binding of lipids to membrane proteins (Gupta et al, 2018;Laganowsky et al, 2014). Coupling this approach to a mutagenesis strategy could provide an exciting route to identify putative cholesterol interaction regions.…”