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Mass spectrometry of intact membrane protein complexes

Nature Protocols volume 8, pages 639–651 (2013)Cite this article

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Abstract

Mass spectrometry (MS) of intact soluble protein complexes has emerged as a powerful technique to study the stoichiometry, structure-function and dynamics of protein assemblies. Recent developments have extended this technique to the study of membrane protein complexes, where it has already revealed subunit stoichiometries and specific phospholipid interactions. Here we describe a protocol for MS of membrane protein complexes. The protocol begins with the preparation of the membrane protein complex, enabling not only the direct assessment of stoichiometry, delipidation and quality of the target complex but also the evaluation of the purification strategy. A detailed list of compatible nonionic detergents is included, along with a protocol for screening detergents to find an optimal one for MS, biochemical and structural studies. This protocol also covers the preparation of lipids for protein-lipid binding studies and includes detailed settings for a quadrupole time-of-flight (Q-TOF) mass spectrometer after the introduction of complexes from gold-coated nanoflow capillaries.

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Figure 1: An overview of a typical membrane protein purification and preparation, as well as analysis by mass spectrometry of the intact complex.
Figure 2: Schematic of a Q-TOF mass spectrometer used for mass measurements of intact membrane protein complexes.
Figure 3: Optimization scheme of critical mass spectrometer parameters for membrane protein native MS.
Figure 4: Effect of ammonium acetate and detergent concentration on mass spectra of the E. coli ammonium channel AmtB.
Figure 5: Additional purification steps lead to an improvement in the overall quality of mass spectra for AmtB.
Figure 6: Mass spectrum activation series for selected detergents from a detergent screen on AmtB-GFP.
Figure 7: Monitoring phsopholipid binding and stoichiometry.

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Acknowledgements

We thank R. Dutzler and I. Zimmerman (University of Zurich) for the ELIC expression plasmid, and D. Rees and C. Gandhi (California Institute of Technology) for the oligomer characterization by addition of mass (OCAM) constructs. The Medical Research Council, European Research Council (ERC) IMPRESS and the Wellcome Trust are acknowledged for funding. A.L. is a Nicholas Kurti Junior Research Fellow of Brasenose College, Oxford and C.V.R. is a Royal Society Professor.

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  1. Department of Chemistry, University of Oxford, Oxford, UK

    Arthur Laganowsky, Eamonn Reading, Jonathan T S Hopper & Carol V Robinson

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  1. Arthur Laganowsky
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Contributions

A.L., E.R., J.T.S.H. and C.V.R. designed the project. A.L. and E.R. performed all experiments, with the exception of the mass spectrometry optimization experiments using ELIC performed by J.T.S.H. A.L. and E.R. prepared the figures. A.L. and C.V.R. wrote the manuscript and coordinated contributions by other authors.

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Correspondence to Carol V Robinson.

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Competing interests

University of Oxford has filed a provisional patent on mass spectrometry mediated drug discovery for membrane proteins using polyoxyethylene glycol detergents.

Supplementary information

Supplementary Methods

Membrane protein expression and purification (PDF 327 kb)

Supplementary Figure 1

Mass spectra dilution series of Ammonium channel, AmtB Purified AmtB in DDM was diluted in Mem MS Buffer containing 2× CMC DDM. Protein concentration of the trimeric complex was determined using a BCA Protein Assay Kit (Pierce, Thermo Scientific) and shown in the upper right corner of each mass spectrum. These results highlight the sensitivity of mass spectrometry. (PDF 571 kb)

Supplementary Figure 2

Mass spectra of the pentameric ligand-gated ion channel from Erwinia chrysanthemi (ELIC) at various purification steps. ELIC was expressed as a HRV3C protease cleavable N-terminal fusion to a maltose binding protein preceded by a secretion signal peptide (pelB) and 10× His-tag22. (a) Mass spectrum of ELIC purified by IMAC and gel filtration chromatography. Peaks are clearly resolved for the pentameric complex and disassociation products poorly resolved. (b) Mass spectrum of ELIC after HRV3C protease treatment and reverse IMAC. Fusion removal has led to increased disassociation products along with resolved spectra all species. (PDF 504 kb)

Supplementary Figure 3

The preparation of lipids can influence membrane protein-lipid interactions. ELIC was titrated with POPE from various preparations to a final concentration of 12.5 μg/mL. Comparison of desodiated (top, purple spectra) and sodiated (middle, blue spectra) lipid preparations prepared as described in BOX 3, and incorrectly using organic solvent, chloroform (bottom, brown spectra). At this resolution desodiated and sodiated lipid preparations are comparable. In contrast, significantly less bound POPE is observed when lipid stocks in chloroform are used. (PDF 339 kb)

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Laganowsky, A., Reading, E., Hopper, J. et al. Mass spectrometry of intact membrane protein complexes. Nat Protoc 8, 639–651 (2013). https://doi.org/10.1038/nprot.2013.024

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