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

Abstract

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