Development of Methodology to Investigate the Surface SMALPome of Mammalian Cells
Extraction of membrane proteins from biological membranes has traditionally involved detergents. In the past decade, a new technique has been developed, which uses styrene maleic acid (SMA) copolymers to extract membrane proteins into nanodiscs without the requirement of detergents. SMA nanodiscs ar...
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| Format: | Article |
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Frontiers Media S.A.
2021-11-01
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| Series: | Frontiers in Molecular Biosciences |
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| Online Access: | https://www.frontiersin.org/articles/10.3389/fmolb.2021.780033/full |
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| author | Kerrie A. Morrison Kerrie A. Morrison Kerrie A. Morrison Kate J. Heesom Karen J. Edler James Doutch Gareth J. Price Gareth J. Price Francoise Koumanov Paul Whitley |
| author_facet | Kerrie A. Morrison Kerrie A. Morrison Kerrie A. Morrison Kate J. Heesom Karen J. Edler James Doutch Gareth J. Price Gareth J. Price Francoise Koumanov Paul Whitley |
| author_sort | Kerrie A. Morrison |
| collection | DOAJ |
| description | Extraction of membrane proteins from biological membranes has traditionally involved detergents. In the past decade, a new technique has been developed, which uses styrene maleic acid (SMA) copolymers to extract membrane proteins into nanodiscs without the requirement of detergents. SMA nanodiscs are compatible with analytical techniques, such as small-angle scattering, NMR spectroscopy, and DLS, and are therefore an attractive medium for membrane protein characterization. While mass spectrometry has also been reported as a technique compatible with copolymer extraction, most studies have focused on lipidomics, which involves solvent extraction of lipids from nanodiscs prior to mass-spectrometry analysis. In this study, mass spectrometry proteomics was used to investigate whether there are qualitative or quantitative differences in the mammalian plasma membrane proteins extracted with SMA compared to a detergent control. For this, cell surface proteins of 3T3L1 fibroblasts were biotinylated and extracted using either SMA or detergent. Following affinity pull-down of biotinylated proteins with NeutrAvidin beads, samples were analyzed by nanoLC-MS. Here, we report for the first time, a global proteomics protocol for detection of a mammalian cell “SMALPome”, membrane proteins incorporated into SMA nanodiscs. Removal of SMA from samples prior to processing of samples for mass spectrometry was a crucial step in the protocol. The reported surface SMALPome of 3T3L1 fibroblasts consists of 205 integral membrane proteins. It is apparent that the detergent extraction method used is, in general, quantitatively more efficient at extracting proteins from the plasma membrane than SMA extraction. However, samples prepared following detergent extraction contained a greater proportion of proteins that were considered to be “non-specific” than in samples prepared from SMA extracts. Tantalizingly, it was also observed that proteins detected uniquely or highly preferentially in pull-downs from SMA extracts were primarily multi-spanning membrane proteins. These observations hint at qualitative differences between SMA and detergent extraction that are worthy of further investigation. |
| first_indexed | 2024-12-13T16:59:20Z |
| format | Article |
| id | doaj.art-ed5fe860386a4b6d918d3a88a6b822fc |
| institution | Directory Open Access Journal |
| issn | 2296-889X |
| language | English |
| last_indexed | 2024-12-13T16:59:20Z |
| publishDate | 2021-11-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Molecular Biosciences |
| spelling | doaj.art-ed5fe860386a4b6d918d3a88a6b822fc2022-12-21T23:37:51ZengFrontiers Media S.A.Frontiers in Molecular Biosciences2296-889X2021-11-01810.3389/fmolb.2021.780033780033Development of Methodology to Investigate the Surface SMALPome of Mammalian CellsKerrie A. Morrison0Kerrie A. Morrison1Kerrie A. Morrison2Kate J. Heesom3Karen J. Edler4James Doutch5Gareth J. Price6Gareth J. Price7Francoise Koumanov8Paul Whitley9Department of Biology and Biochemistry, University of Bath, Bath, United KingdomDepartment of Chemistry, University of Bath, Bath, United KingdomCentre for Sustainable Circular Technologies, University of Bath, Bath, United KingdomUniversity of Bristol, Proteomics Facility, Bristol, United KingdomDepartment of Chemistry, University of Bath, Bath, United KingdomRutherford Appleton Laboratory, ISIS Pulsed Neutron and Muon Source, Harwell Oxford, United KingdomDepartment of Chemistry, University of Bath, Bath, United KingdomDepartment of Chemistry, Khalifa University, Abu Dhabi, United Arab EmiratesDepartment for Health, University of Bath, Bath, United KingdomDepartment of Biology and Biochemistry, University of Bath, Bath, United KingdomExtraction of membrane proteins from biological membranes has traditionally involved detergents. In the past decade, a new technique has been developed, which uses styrene maleic acid (SMA) copolymers to extract membrane proteins into nanodiscs without the requirement of detergents. SMA nanodiscs are compatible with analytical techniques, such as small-angle scattering, NMR spectroscopy, and DLS, and are therefore an attractive medium for membrane protein characterization. While mass spectrometry has also been reported as a technique compatible with copolymer extraction, most studies have focused on lipidomics, which involves solvent extraction of lipids from nanodiscs prior to mass-spectrometry analysis. In this study, mass spectrometry proteomics was used to investigate whether there are qualitative or quantitative differences in the mammalian plasma membrane proteins extracted with SMA compared to a detergent control. For this, cell surface proteins of 3T3L1 fibroblasts were biotinylated and extracted using either SMA or detergent. Following affinity pull-down of biotinylated proteins with NeutrAvidin beads, samples were analyzed by nanoLC-MS. Here, we report for the first time, a global proteomics protocol for detection of a mammalian cell “SMALPome”, membrane proteins incorporated into SMA nanodiscs. Removal of SMA from samples prior to processing of samples for mass spectrometry was a crucial step in the protocol. The reported surface SMALPome of 3T3L1 fibroblasts consists of 205 integral membrane proteins. It is apparent that the detergent extraction method used is, in general, quantitatively more efficient at extracting proteins from the plasma membrane than SMA extraction. However, samples prepared following detergent extraction contained a greater proportion of proteins that were considered to be “non-specific” than in samples prepared from SMA extracts. Tantalizingly, it was also observed that proteins detected uniquely or highly preferentially in pull-downs from SMA extracts were primarily multi-spanning membrane proteins. These observations hint at qualitative differences between SMA and detergent extraction that are worthy of further investigation.https://www.frontiersin.org/articles/10.3389/fmolb.2021.780033/fullstyrene maleic acidSMAmass spectrometry proteomicsSMALPSMALPome |
| spellingShingle | Kerrie A. Morrison Kerrie A. Morrison Kerrie A. Morrison Kate J. Heesom Karen J. Edler James Doutch Gareth J. Price Gareth J. Price Francoise Koumanov Paul Whitley Development of Methodology to Investigate the Surface SMALPome of Mammalian Cells Frontiers in Molecular Biosciences styrene maleic acid SMA mass spectrometry proteomics SMALP SMALPome |
| title | Development of Methodology to Investigate the Surface SMALPome of Mammalian Cells |
| title_full | Development of Methodology to Investigate the Surface SMALPome of Mammalian Cells |
| title_fullStr | Development of Methodology to Investigate the Surface SMALPome of Mammalian Cells |
| title_full_unstemmed | Development of Methodology to Investigate the Surface SMALPome of Mammalian Cells |
| title_short | Development of Methodology to Investigate the Surface SMALPome of Mammalian Cells |
| title_sort | development of methodology to investigate the surface smalpome of mammalian cells |
| topic | styrene maleic acid SMA mass spectrometry proteomics SMALP SMALPome |
| url | https://www.frontiersin.org/articles/10.3389/fmolb.2021.780033/full |
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