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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Autores principales:	Kerrie A. Morrison, Kate J. Heesom, Karen J. Edler, James Doutch, Gareth J. Price, Francoise Koumanov, Paul Whitley
Formato:	article
Lenguaje:	EN
Publicado:	Frontiers Media S.A. 2021
Materias:	styrene maleic acid SMA mass spectrometry proteomics SMALP SMALPome Biology (General) QH301-705.5
Acceso en línea:	https://doaj.org/article/ed5fe860386a4b6d918d3a88a6b822fc
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spelling	oai:doaj.org-article:ed5fe860386a4b6d918d3a88a6b822fc2021-11-18T07:57:14ZDevelopment of Methodology to Investigate the Surface SMALPome of Mammalian Cells2296-889X10.3389/fmolb.2021.780033https://doaj.org/article/ed5fe860386a4b6d918d3a88a6b822fc2021-11-01T00:00:00Zhttps://www.frontiersin.org/articles/10.3389/fmolb.2021.780033/fullhttps://doaj.org/toc/2296-889XExtraction 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.Kerrie A. MorrisonKerrie A. MorrisonKerrie A. MorrisonKate J. HeesomKaren J. EdlerJames DoutchGareth J. PriceGareth J. PriceFrancoise KoumanovPaul WhitleyFrontiers Media S.A.articlestyrene maleic acidSMAmass spectrometry proteomicsSMALPSMALPomeBiology (General)QH301-705.5ENFrontiers in Molecular Biosciences, Vol 8 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	styrene maleic acid SMA mass spectrometry proteomics SMALP SMALPome Biology (General) QH301-705.5
spellingShingle	styrene maleic acid SMA mass spectrometry proteomics SMALP SMALPome Biology (General) QH301-705.5 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
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	article
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
title	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_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_sort	development of methodology to investigate the surface smalpome of mammalian cells
publisher	Frontiers Media S.A.
publishDate	2021
url	https://doaj.org/article/ed5fe860386a4b6d918d3a88a6b822fc
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Development of Methodology to Investigate the Surface SMALPome of Mammalian Cells

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