Chemical Additives Enable Native Mass Spectrometry Measurement of Membrane Protein Oligomeric State within Intact Nanodiscs

Abstract

Membrane proteins play critical biochemical roles but remain challenging to study. Recently, native or nondenaturing mass spectrometry (MS) has made great strides in characterizing membrane protein interactions. However, conventional native MS relies on detergent micelles, which may disrupt natural interactions. Lipoprotein nanodiscs provide a platform to present membrane proteins for native MS within a lipid bilayer environment, but prior native MS of membrane proteins in nanodiscs has been limited by the intermediate stability of nanodiscs. It is difficult to eject membrane proteins from nanodiscs for native MS but also difficult to retain intact nanodisc complexes with membrane proteins inside. Here, we employed chemical reagents that modulate the charge acquired during electrospray ionization (ESI). By modulating ESI conditions, we could either eject the membrane protein complex with few bound lipids or capture the intact membrane protein nanodisc complex-allowing measurement of membrane protein oligomeric state within an intact lipid bilayer environment. The dramatic differences in the stability of nanodiscs under different ESI conditions opens new applications for native MS of nanodiscs.

Charge Reduction of Membrane Proteins in Native Mass Spectrometry Using Alkali Metal Acetate Salts

“…To overcome these drawbacks, several means to reduce membrane protein charge have been reported. These include instrument modification, solubilization with different detergents, and addition of small molecules (14,15,13,16). Instrument modification is the most effective, though it is prohibitive for many groups (14).…”

Conformational dynamics of α-synuclein during the interaction with phospholipid nanodiscs by Millisecond Hydrogen Deuterium Exchange Mass Spectrometry

“…In the case of NMR, intact nanodiscs were identified via a characteristic sharp peak at 0ppm (80)(81)(82). By ESI-MS, nanodiscs generate a broad 'hump-like' peak resulting from incomplete desolvation and polydispersity in the number of lipids contained in individual nanodiscs ( Figure 2c) (83)(84)(85). Together, these data unambiguously demonstrate successful generation and purification of MSP1D1 H5 nanodiscs.…”

Native mass spectrometry—A valuable tool in structural biology

“…110,111 A first study explored nanodiscs, bicelles and amphipols for native MS and showed that indeed the native oligomeric states of several integral membrane proteins were preserved in nanodiscs and bicelles while they differed from those obtained from detergent micelles or amphipols. 112 In particular, nanodiscs were therefore examined in detail; the complex and heterogeneous mass spectra further induced improvements in spectral deconvolution, [113][114][115][116] sample preparation [117][118][119] and method optimisation. 119,120 These improvements allowed, for instance, monitoring the insertion and oligomer assembly in the lipid bilayer.…”