Quantification of Detergents Complexed with Membrane Proteins

Vincent Chaptal,Charlène Prier,Stéphanie Ravaud,Frédéric Delolme,Cédric Montigny,Cédric Orelle,Anass Jawhari,Martin Picard,Renaud Wagner,Sandrine Magnard,Isabelle Broutin,Olivier Bornert,Morgane Agez,Jean‐Michel Jault ,Arnaud Kilburg,Marc Le Maire,Eva Pebay‐Peyroula ,Luca Monticelli,H Ronald Kaback,Pierre Falson

doi:10.1038/srep41751

Abstract

Most membrane proteins studies require the use of detergents, but because of the lack of a general, accurate and rapid method to quantify them, many uncertainties remain that hamper proper functional and structural data analyses. To solve this problem, we propose a method based on matrix-assisted laser desorption/ionization mass spectrometry (MALDI-TOF MS) that allows quantification of pure or mixed detergents in complex with membrane proteins. We validated the method with a wide variety of detergents and membrane proteins. We automated the process, thereby allowing routine quantification for a broad spectrum of usage. As a first illustration, we show how to obtain information of the amount of detergent in complex with a membrane protein, essential for liposome or nanodiscs reconstitutions. Thanks to the method, we also show how to reliably and easily estimate the detergent corona diameter and select the smallest size, critical for favoring protein-protein contacts and triggering/promoting membrane protein crystallization, and to visualize the detergent belt for Cryo-EM studies.

Highlights

Detergents play a major role in handling membrane proteins
We tested the method with various detergents used for structural and functional biology, including (Fig. 1b) sugar derivatives such as n-dodecyl-β-D-maltoside (DDM), n-octyl-β-D-glucoside (OG) and lauryl maltose neopentyl glycol (LMNG)[17], ionic detergents such as Fos-Choline 12 (FC12) and 3-[(3-cho lamidopropyl)-dimethylammonio]-1-propane sulfonate (CHAPS), anionic detergents such as decyl tris[carboxymethyl]-monoalkoxy-trihydroxycalix[4]arene (C4C10)[18], and bile-type detergents such as CHAPS and cholate
Standards were chosen structurally close for the other detergents: decyl MNG (DMNG) for LMNG, C4C12 for C4C10, 2-hydroxy CHAPS (CHAPSO) for CHAPS and deoxycholate for cholate

Summary

Introduction

Detergents play a major role in handling membrane proteins. They are indispensable tools for extracting membrane proteins from the membrane and maintaining them in a soluble and active state for further study. Other methods developed to measure detergent concentrations include: (i) colorimetric assays to estimate the sugar moiety for specific detergents[5,6]; (ii) Fourier transform Infrared spectroscopy[7]; (iii) plain thin layer chromatography coupled with densitometric quantification, or more recently coupled with laser densitometry[8,9]; drop-shape based quantification[10]; (iv) liquid chromatography/ESI-MS11; (v) size-exclusion chromatography coupled with multi-angle laser light scattering[12,13] and analytical ultracentrifugation[14] Useful, these methods are laborious, difficult to implement routinely, limited to a given type of detergent or inapplicable to detergent mixtures. The method provides a rapid means of assaying the amount of bound detergent surrounding a membrane protein and to estimate the size of the detergent corona

Methods

Results

Conclusion