From bottom-up to cell surface proteomics: detergents or no detergents, that is the question.

Measuring the expression levels of membrane proteins (MPs) is crucial for understanding cell differentiation and tissue specificity, defining disease characteristics, identifying biomarkers, and developing therapeutics. While bottom-up proteomics addresses the need for accurately surveying the membrane proteome, the lower abundance and hydrophobic nature of MPs pose challenges in sample preparation. As MPs normally reside in the lipid bilayer, conventional extraction methods rely on detergents, introducing here a paradox - detergents prevent aggregation and facilitate protein processing, but themselves become contaminants that interfere with downstream analytical applications. Various detergent removal methods exist to mitigate this issue, including filter-aided sample preparation, SP3, suspension trapping, and membrane mimetics. This review delves into the fundamentals of each strategy, applications, merits, and limitations, providing insights into their effectiveness in MP research.

Capture of endogenous lipids in peptidiscs and effect on protein stability and activity.

Compared to protein-protein and protein-nucleic acid interactions, our knowledge of protein-lipid interactions remains limited. This is primarily due to the inherent insolubility of membrane proteins (MPs) in aqueous solution. The traditional use of detergents to overcome the solubility barrier destabilizes MPs and strips away certain lipids that are increasingly recognized as crucial for protein function. Recently, membrane mimetics have been developed to circumvent the limitations. In this study, using the peptidisc, we find that MPs in different lipid states can be isolated based on protein purification and reconstitution methods, leading to observable effects on MP activity and stability. Peptidisc also enables re-incorporating specific lipids to fine-tune the protein microenvironment and assess the impact on downstream protein associations. This study offers a first look at the illusive protein-lipid interaction specificity, laying the path for a systematic evaluation of lipid identity and contributions to membrane protein function.

Affinity Purification of Membrane ß-Barrel Proteins via Biotin-Tagged Peptidiscs.

ß-barrel membrane proteins play a crucial role in bacterial pathogenesis and antibiotic resistance, making them a prime focus for the development of new antibiotics and therapeutics. However, their inherent hydrophobic nature and limited presence pose challenges for their high-throughput characterization using conventional methods. In this context, we present a simple but efficacious approach using peptidisc, a membrane mimetic, to overcome the low abundance and hydrophobicity of these proteins. Our methodology, illustrated here using Escherichia coli (E. coli) as a model organism, covers the entire process from outer membrane fraction preparation to data analysis. This detailed protocol outlines the purification of a diverse collection of ß-barrel membrane proteins, rendering them water-soluble and readily amenable to mass spectrometry and downstream drug screening strategies.

Capture of the Mouse Organ Membrane Proteome Specificity in Peptidisc Libraries.

Membrane proteins, particularly those on the cell surface, play pivotal roles in diverse physiological processes, and their dysfunction is linked to a broad spectrum of diseases. Despite being crucial biomarkers and therapeutic drug targets, their low abundance and hydrophobic nature pose challenges in isolation and quantification, especially when extracted from tissues and organs. To overcome these hurdles, we developed the membrane-mimicking peptidisc, enabling the isolation of the membrane proteome in a water-soluble library conducive to swift identification through liquid chromatography with tandem mass spectrometry. This study applies the method across five mice organs, capturing between 200 and 450 plasma membrane proteins in each case. More than just membrane protein identification, the peptidisc is used to estimate the relative abundance across organs, linking cell-surface protein molecular functions to organ biological roles, thereby contributing to the ongoing discourse on organ specificity. This contribution holds substantial potential for unveiling new avenues in the exploration of biomarkers and downstream applications involving knowledge of the organ cell-surface proteome.

A Peptidisc-Based Survey of the Plasma Membrane Proteome of a Mammalian Cell.

Membrane proteins play critical roles at the cell surface and their misfunction is a hallmark of many human diseases. A precise evaluation of the plasma membrane proteome is therefore essential for cell biology and for discovering novel biomarkers and therapeutic targets. However, the low abundance of this proteome relative to soluble proteins makes it difficult to characterize, even with the most advanced proteomics technologies. Here, we apply the peptidisc membrane mimetic to purify the cell membrane proteome. Using the HeLa cell line as a reference, we capture 500 different integral membrane proteins, with half annotated to the plasma membrane. Notably, the peptidisc library is enriched with several ABC, SLC, GPCR, CD, and cell adhesion molecules that generally exist at low to very low copy numbers in the cell. We extend the method to compare two pancreatic cell lines, Panc-1 and hPSC. Here we observe a striking difference in the relative abundance of the cell surface cancer markers L1CAM, ANPEP, ITGB4, and CD70. We also identify two novel SLC transporters, SLC30A1 and SLC12A7, that are highly present in the Panc-1 cell only. The peptidisc library thus emerges as an effective way to survey and compare the membrane proteome of mammalian cells. Furthermore, since the method stabilizes membrane proteins in a water-soluble state, members of the library, here SLC12A7, can be specifically isolated.

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

The outer membrane of Gram-negative bacteria plays a critical role in protecting the cell against external stressors, including antibiotics, and therefore is a prime target for antimicrobial discovery. To facilitate the discovery efforts, a precise knowledge of the outer membrane proteome, and possible variations during pathogenesis, is important. Characterization of the bacterial outer membrane remain challenging, however, and low throughput, due to the high hydrophobicity and relatively low abundance of this cell compartment. Here we adapt our peptidisc-based method to selectively isolate the outer membrane proteome before analysis by mass spectrometry. Using a dual detergent membrane solubilization approach, followed by protein purification in peptidiscs, we capture over 70 outer membrane proteins, including 26 integral ß-barrels and 26 lipoproteins. Many of these proteins are present at high peptide intensities, indicative of a high abundance in the library sample. We further show that the isolated outer membrane proteome can be employed in downstream ligand-binding assays. This peptidisc library made of outer membrane proteins may therefore be useful to systematically survey other bacterial outer membrane proteomes, but also as a nanoparticle format able to support the discovery of next-generation antimicrobials. Data are available via ProteomeXchange identifier PXD036749.

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

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.

Structural Dynamics of the Functional Nonameric Type III Translocase Export Gate.

Type III protein secretion is widespread in Gram-negative pathogens. It comprises the injectisome with a surface-exposed needle and an inner membrane translocase. The translocase contains the SctRSTU export channel enveloped by the export gate subunit SctV that binds chaperone/exported clients and forms a putative ante-chamber. We probed the assembly, function, structure and dynamics of SctV from enteropathogenic E. coli (EPEC). In both EPEC and E. coli lab strains, SctV forms peripheral oligomeric clusters that are detergent-extracted as homo-nonamers. Membrane-embedded SctV9 is necessary and sufficient to act as a receptor for different chaperone/exported protein pairs with distinct C-domain binding sites that are essential for secretion. Negative staining electron microscopy revealed that peptidisc-reconstituted His-SctV9 forms a tripartite particle of ∼22 nm with a N-terminal domain connected by a short linker to a C-domain ring structure with a ∼5 nm-wide inner opening. The isolated C-domain ring was resolved with cryo-EM at 3.1 Å and structurally compared to other SctV homologues. Its four sub-domains undergo a three-stage "pinching" motion. Hydrogen-deuterium exchange mass spectrometry revealed this to involve dynamic and rigid hinges and a hyper-flexible sub-domain that flips out of the ring periphery and binds chaperones on and between adjacent protomers. These motions are coincident with local conformational changes at the pore surface and ring entry mouth that may also be modulated by the ATPase inner stalk. We propose that the intrinsic dynamics of the SctV protomer are modulated by chaperones and the ATPase and could affect allosterically the other subunits of the nonameric ring during secretion.

Structural and functional diversity calls for a new classification of ABC transporters.

Members of the ATP-binding cassette (ABC) transporter superfamily translocate a broad spectrum of chemically diverse substrates. While their eponymous ATP-binding cassette in the nucleotide-binding domains (NBDs) is highly conserved, their transmembrane domains (TMDs) forming the translocation pathway exhibit distinct folds and topologies, suggesting that during evolution the ancient motor domains were combined with different transmembrane mechanical systems to orchestrate a variety of cellular processes. In recent years, it has become increasingly evident that the distinct TMD folds are best suited to categorize the multitude of ABC transporters. We therefore propose a new ABC transporter classification that is based on structural homology in the TMDs.

Correction to: A Mathematical Model for the Kinetics of the MalFGK2 Maltose Transporter.

The original version of this article unfortunately contained a mistake. The co-author Dr. Franck Duong Van Hoa first name and last name were misinterpreted in the original publication.

His-Tagged Peptidiscs Enable Affinity Purification of the Membrane Proteome for Downstream Mass Spectrometry Analysis.

Characterization of the integral membrane proteome by mass spectrometry (MS) remains challenging due its high complexity and inherent insolubility. In a typical experiment, the cellular membranes are isolated, the proteins are solubilized and fractionated, and the detergent micelles are removed before MS analysis. Detergents are not compatible with mass spectrometry, however, and their removal from biological samples often results in reduced protein identification. As an alternative to detergents, we recently developed the peptidisc membrane mimetic, which allows entrapment of the cell envelope proteome into water-soluble particles, termed a "peptidisc library". Here, we employ a His-tagged version of the peptidisc peptide scaffold to enrich the reconstituted membrane proteome by affinity chromatography. This purification step reduces the sample complexity by depleting ribosomal and soluble proteins that often cosediment with cellular membranes. As a result, the peptidisc library is enriched in low-abundance membrane proteins. We apply this method to survey changes in the membrane proteome upon depletion of the SecDFyajC complex, the ancillary subunit of the Sec translocon. In the depleted strain, we detect increased membrane localization of the motor ATPase SecA, along with increased levels of an unannotated inner membrane protein, YibN. Together, these results demonstrate the utility of the peptidisc for global purification of membrane proteins and for monitoring change in the membrane proteome.

New approach for membrane protein reconstitution into peptidiscs and basis for their adaptability to different proteins.

Previously we introduced peptidiscs as an alternative to detergents to stabilize membrane proteins in solution (Carlson et al., 2018). Here, we present 'on-gradient' reconstitution, a new gentle approach for the reconstitution of labile membrane-protein complexes, and used it to reconstitute Rhodobacter sphaeroides reaction center complexes, demonstrating that peptidiscs can adapt to transmembrane domains of very different sizes and shapes. Using the conventional 'on-bead' approach, we reconstituted Escherichia coli proteins MsbA and MscS and find that peptidiscs stabilize them in their native conformation and allow for high-resolution structure determination by cryo-electron microscopy. The structures reveal that peptidisc peptides can arrange around transmembrane proteins differently, thus revealing the structural basis for why peptidiscs can stabilize such a large variety of membrane proteins. Together, our results establish the gentle and easy-to-use peptidiscs as a potentially universal alternative to detergents as a means to stabilize membrane proteins in solution for structural and functional studies.

PeptiQuick, a One-Step Incorporation of Membrane Proteins into Biotinylated Peptidiscs for Streamlined Protein Binding Assays.

Membrane proteins, including transporters, channels, and receptors, constitute nearly one-fourth of the cellular proteome and over half of current drug targets. Yet, a major barrier to their characterization and exploitation in academic or industrial settings is that most biochemical, biophysical, and drug screening strategies require these proteins to be in a water-soluble state. Our laboratory recently developed the peptidisc, a membrane mimetic offering a "one-size-fits-all" approach to the problem of membrane protein solubility. We present here a streamlined protocol that combines protein purification and peptidisc reconstitution in a single chromatographic step. This workflow, termed PeptiQuick, allows for bypassing dialysis and incubation with polystyrene beads, thereby greatly reducing exposure to detergent, protein denaturation, and sample loss. When PeptiQuick is performed with biotinylated scaffolds, the preparation can be directly attached to streptavidin-coated surfaces. There is no need to biotinylate or modify the membrane protein target. PeptiQuick is showcased here with the membrane receptor FhuA and antimicrobial ligand colicin M, using biolayer interferometry to determine the precise kinetics of their interaction. It is concluded that PeptiQuick is a convenient way to prepare and analyze membrane protein-ligand interactions within one day in a detergent-free environment.

Profiling the Escherichia coli membrane protein interactome captured in Peptidisc libraries.

Protein-correlation-profiling (PCP), in combination with quantitative proteomics, has emerged as a high-throughput method for the rapid identification of dynamic protein complexes in native conditions. While PCP has been successfully applied to soluble proteomes, characterization of the membrane interactome has lagged, partly due to the necessary use of detergents to maintain protein solubility. Here, we apply the peptidisc, a 'one-size fits all' membrane mimetic, for the capture of the Escherichia coli cell envelope proteome and its high-resolution fractionation in the absence of detergent. Analysis of the SILAC-labeled peptidisc library via PCP allows generation of over 4900 possible binary interactions out of >700,000 random associations. Using well-characterized membrane protein systems such as the SecY translocon, the Bam complex and the MetNI transporter, we demonstrate that our dataset is a useful resource for identifying transient and surprisingly novel protein interactions. For example, we discover a trans-periplasmic supercomplex comprising subunits of the Bam and Sec machineries, including membrane-bound chaperones YfgM and PpiD. We identify RcsF and OmpA as bone fide interactors of BamA, and we show that MetQ association with the ABC transporter MetNI depends on its N-terminal lipid anchor. We also discover NlpA as a novel interactor of MetNI complex. Most of these interactions are largely undetected by standard detergent-based purification. Together, the peptidisc workflow applied to the proteomic field is emerging as a promising novel approach to characterize membrane protein interactions under native expression conditions and without genetic manipulation.