Development of Methodology to Investigate the Surface SMALPome of Mammalian Cells

Abstract

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.

Lipid vesicle formation by encapsulation of SMALPs in surfactant-stabilised droplets

“…Ca 2+ has been reported to induce polymer aggregation in literature. 27,42,43 Visible aggregation of lipid structures occurred after Ca 2+ addition. This led to the disruption of some GUVs; nevertheless, a subset of GUVs remained intact.…”

The bigger picture: global analysis of solubilization performance of classical detergents versus new synthetic polymers utilizing shotgun proteomics

“…For most of these polymers, high-resolution Cryo-EM structures are available for membrane proteins solubilized from bacterial (Catalano et al, 2021;Doyle et al, 2022;Flegler et al, 2020;Higgins et al, 2021;Qiu et al, 2018;Sun et al, 2018;Swainsbury et al, 2023;Tascón et al, 2020) or eukaryotic cells (Yoder and Gouaux, 2020;Yu et al, 2021), many of which showing densities consistent with the presence of native lipids (Catalano et al, 2021;Doyle et al, 2022;Flegler et al, 2020;Qiu et al, 2018;Yoder and Gouaux, 2020). Unfortunately, while there is clear evidence that all these polymers can form native nanodiscs, most of the information about their solubilization capabilities comes from selected example proteins and systematic characterization of their solubilization capacity against other methods has only recently been performed for SMA (Carlson et al, 2019;Morrison et al, 2021).…”