Membrane Mimetic-Thermal Proteome Profiling Reveals Broad, Sequence-Independent Membrane Protein Stabilization by Cholesteryl Hemisuccinate

Membrane protein stability is strongly influenced by the surrounding lipid environment, yet how individual lipid species shape membrane proteome stability remains poorly understood. Here, we systematically examined the impact of sphingomyelin, 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), and cholesteryl hemisuccinate (CHS) on membrane proteomes using membrane mimetic platforms combined with membrane mimetic thermal proteome profiling (MM-TPP). CHS shifted the proteome composition away from soluble proteins and toward integral membrane proteins, and induced concentration-dependent thermal stabilization of the mouse liver membrane proteome. Organellar membrane proteins, which displayed greater intrinsic lability than plasma membrane proteins, showed preferential stabilization by CHS. CHS supplementation of E. coli membranes similarly produced broad stabilization, indicating that this effect occurs even in cholesterol-naive systems. CHS responses were reproducible across Peptidisc and DDM and independent of CRAC/CARC motif density, supporting a broad, sequence-independent mechanism rather than selective lipid binding, although stabilization was greater among proteins with more transmembrane helices. Accordingly, individual purified proteins reconstituted with CHS exhibited only modest stabilization, consistent with a broad effect that is more apparent at the proteome scale than for any single protein examined in isolation. Together, these findings redefine CHS as a general sterol scaffold that broadly stabilizes membrane proteins and establish MM-TPP as a versatile platform for investigating lipid-dependent effects on membrane proteome stability. Subject areaIntegral Membrane Proteins, Thermal Proteome Profiling, Membrane Mimetics, Cholesterol, Lipid-Protein Interactions, Mass Spectrometry HighlightsO_LICHS broadly stabilizes membrane proteins across diverse membrane mimetics. C_LIO_LIOrganellar membrane proteins exhibit the strongest CHS-mediated stabilization. C_LIO_LICHS stabilization is conserved in cholesterol-naive E. coli. C_LIO_LIMM-TPP enables proteome-wide analysis of lipid-dependent protein stability. C_LI Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=71 SRC="FIGDIR/small/745344v1_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@1a3e017org.highwire.dtl.DTLVardef@1cd4464org.highwire.dtl.DTLVardef@1453da7org.highwire.dtl.DTLVardef@d2dc1d_HPS_FORMAT_FIGEXP M_FIG C_FIG