Development of a Method Combining Peptidiscs and Proteomics to Identify, Stabilize, and Purify a Detergent-Sensitive Membrane Protein Assembly

Abstract

The peptidisc membrane mimetic enables global reconstitution of the bacterial membrane proteome into water-soluble detergent-free particles, termed peptidisc libraries. We present here a method that combines peptidisc libraries and chromosomal-level gene tagging technology with affinity purification and mass spectrometry (AP/MS) to stabilize and identify fragile membrane protein complexes that exist at native expression levels. This method circumvents common artifacts caused by bait protein overproduction and protein complex dissociation due to lengthy exposure to detergents during protein isolation. Using the Escherichia coli Sec system as a case study, we identify an expanded version of the translocon, termed the HMD complex, consisting of nine different integral membrane subunits. This complex is stable in peptidiscs but dissociates in detergents. Guided by this native-level proteomic information, we design and validate a procedure that enables purification of the HMD complex with minimal protein dissociation. These results highlight the utility of peptidiscs and AP/MS to discover and stabilize fragile membrane protein assemblies. Data are available via ProteomeXchange with identifier PXD032315.

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

“…This technique has been acknowledged for its ability to isolate low-solubility aggregates that often include insoluble transmembrane proteins [40]. On the other hand, the peptidisc captures transmembrane proteins in a soluble state using a His 6 -tagged scaffold that enables their selective recovery through Ni-NTA chromatography [41,42]. Despite the distinct approaches, the methods share a common initial step of ultracentrifugation to enrich the membrane fraction and deplete high-abundance soluble proteins that often obscure membrane protein detection [43].…”

A Dual Detergent Strategy to Capture a Bacterial Outer Membrane Proteome in Peptidiscs for Characterization by Mass Spectrometry and Binding Assays

“…We recently reported the peptidisc as a useful tool for interrogating the protein composition of the Gram-negative bacteria cell envelope by mass spectrometry. ,, We focused on inner membrane protein complexesparticularly the universally conserved Sec translocon . In this paper, we extend this peptidisc-based approach to isolate the outer membrane proteome, which includes both β-barrel proteins and outer membrane lipoproteins.…”

“…18,19,21 We focused on inner membrane protein complexes�particularly the universally conserved Sec translocon. 21 In this paper, we extend this peptidisc-based approach to isolate the outer membrane proteome, which includes both β-barrel proteins and outer membrane lipoproteins. This approach is based on a 2-step, "Triton-then-LDAO", detergent extraction strategy to solubilize the outer membrane proteome preferentially, followed by reconstitution in biotinylated peptidiscs and purification over Streptavidin Mutein Matrix resin.…”

“…Unlysed cells were removed by centrifugation (6000 g , 10 min, 4 °C). Cellular membranes were pelleted by ultracentrifugation (200 000 g , 30 min, 4 °C), then resuspended in Buffer A, and reconstituted into peptidiscs as previously described with minor modifications. ,, Briefly, membranes (500 μL at 2 mg/mL) were solubilized with 0.5% DDM for 30 min with gentle shaking at 4 °C. The sample was ultracentrifuged at ∼200 000 g (30 min, 4 °C) and the supernatant was collected.…”

Nanodisc reconstitution and characterization of amyloid-β precursor protein C99

“…Although detergents are cost-effective and efficient in extracting membrane proteins, they have limitations, as their short alkyl chains and headgroups are chemically distinct from those of native membrane lipids, rendering them discrete physicochemical properties that can affect the function of membrane proteins (5,(11)(12)(13). Moreover, their denaturing properties make it difficult to maintain membrane protein's oligomeric or multimeric states that are stabilized by intermolecular interactions (14)(15)(16)(17). Planar lipid-bilayer-containing bicelles have been used extensively to study the structure, dynamics, and membrane topology of a variety of membrane proteins (18).…”