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

ABSTRACT: The low abundance and hydrophobicity of plasma membrane proteome relative to soluble proteins makes it difficult to characterize. Here, we apply the peptidisc membrane mimetic to purify the cell membrane proteome. We used HeLa cells as a reference to establish the method, and compared the plasma membrane proteomes between Pancreatic cancer cell Panc-1 and hPSC.

INSTRUMENT(S): LTQ Orbitrap Elite

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Zhiyu Zhao

LAB HEAD: Franck Duong

PROVIDER: PXD039990 | Pride | 2023-10-24

REPOSITORIES: Pride

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			Action	DRS
	20201016_Lumos_CDS_ZhaoK_HeLa_DDM.index	Other
	20201016_Lumos_CDS_ZhaoK_HeLa_DDM.raw	Raw
	20201016_Lumos_CDS_ZhaoK_HeLa_His_Lib.index	Other
	20201016_Lumos_CDS_ZhaoK_HeLa_His_Lib.raw	Raw
	20210108_UVPD_CDS_ZhaoK_HeLa_His-E.index	Other

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

Zhao Zhiyu Z Khurana Arshdeep A Antony Frank F Young John W JW Hewton Keeley G KG Brough Zora Z Zhong Tianshuang T Parker Seth J SJ Duong van Hoa Franck F

Molecular & cellular proteomics : MCP 20230607 8

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 ...[more]

PMID: 37295717

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

Project description:The low abundance and hydrophobicity of plasma membrane proteome relative to soluble proteins makes it difficult to characterize. Here, we apply the peptidisc membrane mimetic to purify the cell membrane proteome. We used HeLa cells as a reference to establish the method, and compared the plasma membrane proteomes between Pancreatic cancer cell Panc-1 and hPSC.

2023-10-24 | PXD041913 | Pride

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

Project description: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 E. coli Sec system as a case study, we identify an expanded version of the translocon, termed the HMD complex, consisting of 9 different integral membrane subunits. This complex is stable in peptidiscs but dissociates in detergent. 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.

2022-06-09 | PXD032315 | Pride

A Simple Method to Isolate and Characterize a Bacterial Outer Membrane Proteome by Bottom-Up Mass Spectrometry

Project description:The outer membrane of gram-negative bacteria plays a critical role in protecting the cell against external stressors, including antibiotics. Given its importance to the resistance mechanisms, the outer membrane is a prime target for antimicrobial drug discovery. To facilitate discovery efforts, however, a precise characterization of the outer membrane protein content – or the "outer membrane proteome" - and possible variations during bacterial resistance, is important. This information is necessary to understand how bacteria interact with their host organism during infection. However, proteomic-based investigations of the bacterial outer membrane remain technically challenging, given this cell compartment's lower abundance and high hydrophobicity. We report here a simple but efficacious enrichment of the bacterial outer membrane proteome for downstream characterization by mass spectrometry. We start with a dual detergent approach to preferentially solubilize the outer membrane, followed by reconstitution in peptidisc library to isolate and purify the entire outer membrane proteome at once. Our method identifies 71 outer membrane proteins (OMPs), including 26 integral -barrels and 26 lipoproteins; many of which are present with high-intensity and peptide number values, indicative of a high abundance in the library sample. Given this enrichment, the method may be useful for comparing outer membrane proteomes. We also show that the library can also be employed for downstream protein binding assays.

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Project description:We recently presented the peptidisc membrane mimetic for the global reconstitution of membrane proteomes into water-soluble detergent-free particles, termed peptidisc library. We here develop a method that combines the peptidisc library with affinity pulldown and mass spectrometry (AP/MS) to explore the membrane interactome at native expression level, which minimizes artifacts due to protein overproduction or exposure to detergents. Using the Sec translocon as a case study, we identify an expanded holo-translocon complex, termed the HMD complex, which consists of 9 different membrane subunits - SecYEG, SecDFyajC, YidC, plus YfgM and PpiD. This super-complex can be biochemically isolated in peptidisc but not in detergent. We also detect associations of the HMD complex with the outer membrane Bam complex and we discover a novel unannotated inner membrane protein named YibN. This interactome analysis at native expression level points out the membrane complexes that are dissociated in detergent, yet stable enough to be captured in peptidiscs. We believe this information is critical for the design of biochemical procedures that will enable their successful purification.

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Expression profiling of NM1 KO and WT mice

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