Detergent-free Lipodisq Nanoparticles Facilitate High-Resolution Mass Spectrometry of Folded Integral Membrane Proteins.

Kin Kuan Hoi,Hsin-Yung Yen,Garrick F Taylor,Peter J Judge,Carol V Robinson,Di Wu,Juan Francisco Bada Juarez,Anthony Watts,Javier Vinals

doi:10.1021/acs.nanolett.0c04911

Abstract

Integral membrane proteins pose considerable challenges to mass spectrometry (MS) owing to the complexity and diversity of the components in their native environment. Here, we use native MS to study the post-translational maturation of bacteriorhodopsin (bR) and archaerhodopsin-3 (AR3), using both octyl-glucoside detergent micelles and lipid-based nanoparticles. A lower collision energy was required to obtain well-resolved spectra for proteins in styrene-maleic acid copolymer (SMA) Lipodisqs than in membrane scaffold protein (MSP) Nanodiscs. By comparing spectra of membrane proteins prepared using the different membrane mimetics, we found that SMA may favor selective solubilization of correctly folded proteins and better preserve native lipid interactions than other membrane mimetics. Our spectra reveal the correlation between the post-translation modifications (PTMs), lipid-interactions, and protein-folding states of bR, providing insights into the process of maturation of the photoreceptor proteins.

Highlights

Integral membrane proteins account for around a quarter of the human proteome[1] and represent approximately 60% of all known drug targets.[2]
■ RESULTS AND DISCUSSION Native Mass Spectra of Protein Lipodisqs Are Well Resolved. bR reconstituted in Lipodisqs, prepared without the use of detergent, or in proteoliposomes were characterized by dynamic light scattering, by circular dichroism, and by their absorbance spectra (Figure S1)
We have demonstrated that native mass spectrometry (MS) spectra of photoreceptor proteins, extracted from native membranes using styrene-maleic acid copolymer (SMA) polymer, are well resolved and that both covalent and noncovalent adducts can be readily identified. bR and AR3 are released from Lipodisq nanoparticles at lower collison voltages compared to membrane scaffold protein (MSP) Nanodiscs

Summary

Introduction

Integral membrane proteins account for around a quarter of the human proteome[1] and represent approximately 60% of all known drug targets.[2]. Complex synthesis and folding pathways are required for these proteins, because the nascent polypeptide chain that emerges from the ribosome must be threaded through the membrane before the final native conformation is achieved. Nanoelectrospray ionization (nESI) enables protein mass spectra to be acquired while maintaining native conformations under non-denaturing conditions.[3,4] Soluble and membraneembedded proteins have been studied using native mass spectrometry (MS), allowing interrogation of post-translational modifications (PTMs), ligand interactions, oligomeric states, and associated lipids.[5,6] For example, native MS results revealed the regulatory role of PTMs on the ligand affinity of glycoproteins.[7] Recent studies have shown that lipids can stabilize the dimer interface(s) of a range of membrane proteins.[8,9]

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