Mass spectrometry of intact membrane protein complexes
Open accessAbstract
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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Membrane proteins generally require nonionic surfactants such as DDM or oligoglycerol detergents for native MS analysis.
“Due to the hydrophobic nature of membrane proteins, the use of nonionic surfactants, such as DDM or the recently developed oligoglycerol class of detergents (shown to improve purification and MS-based characterization of G-protein coupled receptors) [47], to solubilize and stabilize membrane proteins is generally required to enable native MS analysis [48–50].”
From Top-down proteomics: challenges, innovations, and applications in basic and clinical research (2020) · cites this paper as [49]
In native MS of protein-micelle complexes, the complex is ionized and the protein is liberated from the surfactant micelle using high collisional energy.
“In this case, the entire protein-micelle complex is ionized, and the protein is liberated from the surfactant micelle using high collisional energy in the mass spectrometer [49].”
From Top-down proteomics: challenges, innovations, and applications in basic and clinical research (2020) · cites this paper as [49]
Nonionic detergents such as DDM are generally considered MS-compatible and have been used to maintain higher-order structures of membrane protein complexes in native MS.
“Nonionic detergents such as n-Dodecyl-β-D-Maltoside (DDM) have been generally considered MS-compatible, and have even been used for maintaining higher-order structures of membrane protein complexes in native MS.21”
From Sensitive Top-Down Proteomics Analysis of a Low Number of Mammalian Cells Using a Nanodroplet Sample Processing Platform (2020) · cites this paper as [21]
Complex-down analysis has been extended to membrane proteins by adding detergents or lipids in solution to prevent protein precipitation and preserve native structure.
“Recently, this technique has been extended to membrane proteins by adding detergents or lipids in the solution to prevent protein precipitation and to preserve their native structure.”
From Higher-order structural characterisation of native proteins and complexes by top-down mass spectrometry (2020) · cites this paper as [108]
Carefully tuned collisional activation is used to eject intact membrane protein complexes from detergent micelles so that the membrane protein can be efficiently analysed.
“Carefully tuned collisional activation is used to eject intact membrane protein complexes from detergent micelles so that the membrane protein can be efficiently analysed.108”
From Higher-order structural characterisation of native proteins and complexes by top-down mass spectrometry (2020) · cites this paper as [108]
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- Authors
- Laganowsky, A., Reading, E., Hopper, J. T. S., & Robinson, C. V.
- Affiliations
- University of Oxford (GB)
- Corresponding author
- Robinson, Carol V
- Venue
- Nature Protocols
- Published
- 2013-03-07
- Volume
- 8
- Issue
- 4
- Pages
- 639-651
- ISSN
- 1750-2799 (ISSN-L)
- 1754-2189
- PMC
- PMC4058633
- OpenAlex
- W2030533925
- Cited by
- 409
- References
- 25
- Citations by year
- 2026: 14 · 2025: 22 · 2024: 35 · 2023: 33 · 2022: 26 · 2021: 34 · 2020: 42 · 2019: 33 · 2018: 33 · 2017: 52 · 2016: 31 · 2015: 20 · 2014: 25 · 2013: 7
- FWCI
- 12.45
- Type
- journal article
- Language
- en
- Open access
- green
- www.ncbi.nlm.nih.gov
- Licence
- www.springer.com/tdm (tdm)
- Locations
- Nature Protocols (publishedVersion)
- PubMed (publishedVersion)
- Europe PMC (PubMed Central) (acceptedVersion)
- PubMed Central (acceptedVersion)
- ePrints Soton (University of Southampton) (submittedVersion)
- Funders
- Wellcome Trust (088150)
- Topics
- Physical Sciences › Chemistry › Spectroscopy › Mass Spectrometry Techniques and Applications
- Physical Sciences › Chemistry › Spectroscopy › Advanced Proteomics Techniques and Applications
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Lipid Membrane Structure and Behavior
- Keywords
- membrane protein complexes, subunit stoichiometry, delipidation, nonionic detergents, detergent screening, phospholipid interactions, protein-lipid binding, quadrupole time-of-flight mass spectrometry, protein complex purification
- MeSH terms
- Detergents, Indicators and Reagents, Membrane Proteins, Phospholipids, Protein Binding, Mass Spectrometry, Protein Structure, Tertiary
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