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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...
Autores principales: | , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Frontiers Media S.A.
2021
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8637157/ https://www.ncbi.nlm.nih.gov/pubmed/34869600 http://dx.doi.org/10.3389/fmolb.2021.780033 |
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author | Morrison, Kerrie A. Heesom, Kate J. Edler, Karen J. Doutch, James Price, Gareth J. Koumanov, Francoise Whitley, Paul |
author_facet | Morrison, Kerrie A. Heesom, Kate J. Edler, Karen J. Doutch, James Price, Gareth J. Koumanov, Francoise Whitley, Paul |
author_sort | Morrison, Kerrie A. |
collection | PubMed |
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. |
format | Online Article Text |
id | pubmed-8637157 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2021 |
publisher | Frontiers Media S.A. |
record_format | MEDLINE/PubMed |
spelling | pubmed-86371572021-12-03 Development of Methodology to Investigate the Surface SMALPome of Mammalian Cells Morrison, Kerrie A. Heesom, Kate J. Edler, Karen J. Doutch, James Price, Gareth J. Koumanov, Francoise Whitley, Paul Front Mol Biosci Molecular Biosciences 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. Frontiers Media S.A. 2021-11-18 /pmc/articles/PMC8637157/ /pubmed/34869600 http://dx.doi.org/10.3389/fmolb.2021.780033 Text en Copyright © 2021 Morrison, Heesom, Edler, Doutch, Price, Koumanov and Whitley. https://creativecommons.org/licenses/by/4.0/This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. |
spellingShingle | Molecular Biosciences Morrison, Kerrie A. Heesom, Kate J. Edler, Karen J. Doutch, James Price, Gareth J. Koumanov, Francoise Whitley, Paul Development of Methodology to Investigate the Surface SMALPome of Mammalian Cells |
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 | Molecular Biosciences |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8637157/ https://www.ncbi.nlm.nih.gov/pubmed/34869600 http://dx.doi.org/10.3389/fmolb.2021.780033 |
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