Biology subjects

Antony, F.

Publications and source records attributed to Antony, F..

5 recordsLinked to original sources

bioRxiv2026

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

biochemistry↗

bioRxiv2026

A detergent-free workflow for native membrane proteomics using Peptergents

Quantitative membrane proteomics remains fundamentally limited by sample preparation because detergent extraction can perturb membrane protein interactions, ligand-responsive conformations, and higher-order assemblies before mass spectrometric analysis. Here, we demonstrate that peptide-based surfactants (Peptergents) enable a complete detergent-free workflow for native membrane proteomics. Membrane proteins are extracted directly from biological membranes while preserving their structural and functional integrity and remaining fully compatible with downstream LC-MS/MS workflows. Functional preservation is evidenced by maintenance of ligand-responsive conformations in the ABC transporter MsbA and the endogenous GPCR P2RY12, together with stabilization of the detergent-sensitive nine-subunit holo-translocon HTL, indicating that fragile membrane protein assemblies remain intact. At the proteome level, despite recovering fewer membrane proteins than conventional detergent extraction, Peptergent consistently generates higher peptide signal intensities, retains tissue-specific membrane proteome signatures, and preferentially enriches endoplasmic reticulum-associated metabolic networks, including cytochrome P450 enzymes and their interaction network. Together, these findings establish Peptergents as a broadly applicable membrane extraction technology for LC-MS/MS-based membrane proteomics, preserving native membrane organization and expanding the proteomics toolbox for biochemical, structural, and systems-level analyses of membrane proteins. In Brief StatementThis study establishes Peptergents as a detergent-free membrane extraction technology for LC-MS/MS-based membrane proteomics. Peptergent extraction preserves ligand-responsive membrane proteins, fragile membrane protein assemblies, and tissue-specific membrane proteome signatures while remaining fully compatible with quantitative proteomic workflows. These findings provide a broadly applicable strategy for preserving native membrane organization for biochemical, structural, and systems-level analyses of membrane proteins. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=199 SRC="FIGDIR/small/744532v1_ufig1.gif" ALT="Figure 1"> View larger version (56K): org.highwire.dtl.DTLVardef@1cef0c1org.highwire.dtl.DTLVardef@1203aeaorg.highwire.dtl.DTLVardef@fbdb47org.highwire.dtl.DTLVardef@f915e6_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIPeptergents preserve ligand-responsive membrane proteins. C_LIO_LISupport chemoproteomics in thermal proteome profiling assays. C_LIO_LISimplify membrane proteomics workflow. C_LIO_LIMaintain native tissue-specific membrane biology. C_LIO_LIPreserve fragile membrane protein assemblies. C_LI

biochemistry↗

bioRxiv2026

PEPTERGENT: A Peptide-Based Method for Detergent-Free Extraction and Purification of Membrane Proteins and Membrane Proteomes

Peptergent is a novel class of amphipathic peptides that enable detergent-free extraction and purification of membrane proteins (MPs). These designed peptides self-assemble around hydrophobic transmembrane regions of proteins, forming stable, water-soluble assemblies that can be isolated directly from biological membranes. By doing so, Peptergent bypass the limitations imposed by traditional detergents, which often destabilize proteins and restrict downstream analyses. Since detergents are completely avoided, Peptergent-isolated MPs are directly amenable to structural and mass spectrometry (MS) analysis, thereby addressing their persistent underrepresentation in proteomic datasets and improving their accessibility for drug-screening strategies. Here, we describe a streamlined protocol for isolating MPs with the Peptergent PDET-1, followed by exchange into His-tagged Peptidiscs for Ni-NTA-based affinity purification. The method comprises membrane isolation, peptide preparation, protein extraction, clarification, and exchange of MPs from Peptergent to Peptidiscs. Application of this workflow yields enriched membrane proteomes compatible with downstream LC-MS/MS analysis, with improved recovery of hydrophobic and multi-pass membrane proteins. Key featuresO_LIDirect extraction and solubilization of membrane proteins in Peptergents C_LIO_LIExchange into His-tagged Peptidiscs enabling affinity purification of MPs C_LIO_LI100% detergent-free workflow compatible with LC-MS/MS analysis C_LIO_LIApplicable to cultured cells and tissue-derived membrane fractions C_LI In BriefWe describe a Peptergent-based workflow for isolating membrane proteins directly from membrane preparations. Proteins are extracted with the Peptergent peptide scaffold (PDET-1) and transferred into His-tagged Peptidisc (HD-43). The water-soluble membrane proteins are enriched by Ni-NTA affinity purification and prepared for bottom-up mass spectrometry, yielding enriched membrane proteomes and dried peptide samples ready for LC-MS analysis Graphical Overview O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=128 SRC="FIGDIR/small/711971v1_ufig1.gif" ALT="Figure 1"> View larger version (36K): org.highwire.dtl.DTLVardef@2d5bdforg.highwire.dtl.DTLVardef@1eac5fborg.highwire.dtl.DTLVardef@6c14fcorg.highwire.dtl.DTLVardef@1d5d28f_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2025

Comparative Evaluation of Solid-Phase and Membrane Mimetic Strategies in Membrane Proteome Coverage and Disease-State Analysis

Membrane proteins (MPs) are vital to cellular signaling, metabolism, and disease pathology, yet remain underrepresented in proteomics. To address this, several independent workflows have been developed to enable the profiling of the membrane proteome, however the relative advantages and limitations of each method remain poorly defined. Here, we systematically compare four classical solid-phase membrane proteomic workflows (SP3, SP4, FASP, S-Trap) and three membrane mimetic strategies (Peptidisc, nanodisc, and SMALP copolymer) for mass spectrometry-based membrane proteome profiling, using healthy (LFD) and obese (HFD) mouse liver tissue. We found that the solid-phase methods yield higher total protein identifications, while the membrane mimetic systems enrich MPs. SMALP copolymer displays intermediate characteristics between the solid-phase and membrane mimetic workflows. Peptidisc and nanodisc stand out for their enrichment of MPs, although Peptidisc shows better enrichment of plasma membrane integral MPs, particularly those with 11+ transmembrane segments. In the context of HFD-induced liver proteome remodeling, the Peptidisc workflow outperformed the other six methods by capturing the highest number of differentially expressed MPs and demonstrating the greatest accuracy in detecting MP-level dysregulation. Collectively, this comparative analysis highlights the trade-offs between depth of proteome coverage and MP enrichment across workflows, underscoring the importance of method selection based on total protein counts, MP enrichment, and the accurate detection of MP-level dysregulation. HighlightsO_LISystematic comparison of seven workflows for membrane proteomics C_LIO_LISolid-phase methods enrich soluble proteins; mimetics enrich membrane proteins C_LIO_LISMALP displays intermediate performance between other workflows C_LIO_LIPeptidisc captures the most dysregulated membrane proteins in diseased liver C_LIO_LIPeptidisc most accurate in detecting membrane protein dysregulation C_LI In Brief StatementThis study presents a systematic comparison of seven proteomic workflows for membrane protein profiling. Solid-phase methods yield higher total protein identifications, whereas membrane mimetics enrich more membrane proteins. Among tested methods on the diseased mouse liver, Peptidisc captures more differentially expressed membrane proteins and demonstrates superior accuracy in detecting membrane protein-level dysregulation. These findings provide a practical framework for selecting proteomic strategies tailored to membrane protein enrichment and biological insight. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=131 SRC="FIGDIR/small/672181v1_ufig1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@1217dc7org.highwire.dtl.DTLVardef@1c8c188org.highwire.dtl.DTLVardef@119db19org.highwire.dtl.DTLVardef@7f5642_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2024

Profiling the organ membrane proteome dysregulation in the context of liver disease

Alcohol consumption and high-fat diets often coincide in Western society, exerting negative synergistic effects on the liver. While many studies have demonstrated the impact of ALD and NAFLD on organ protein expression, none have offered a comprehensive view of the dysregulation at the level of the membrane proteome. In this study, we utilize peptidisc and solvent precipitation (SP4) methods to isolate and compare the membrane protein content of the liver with its unique biological functions. Using mice treated with a high-fat diet and ethanol in drinking water, we identified 1,563 liver proteins, with 46% predicted to have a transmembrane segment. Among these, 106 integral membrane proteins are dysregulated compared to the untreated sample. Gene ontology analysis reveals several dysregulated membrane processes associated with lipid metabolism, cell adhesion, xenobiotic processing, and mitochondrial membrane formation. Pathways related to cholesterol and bile acid transport are also mutually affected, suggesting an adaptive mechanism to counter the steatosis of the liver model. Our peptidisc-based membrane proteome profiling thus emerges as an effective way to gain insights into the role of the transmembrane proteome in disease development, warranting further in-depth analysis of the individual effect of the identified dysregulated membrane proteins.

systems biology↗