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Biomarkers : biochemical indicators of exposure, response, and susceptibility to chemicals2016; 20(8); 572-589; doi: 10.3109/1354750X.2015.1130191

Label-free proteomic analysis of the hydrophobic membrane protein complement in articular chondrocytes: a technique for identification of membrane biomarkers.

Abstract: There is insufficient knowledge about the chondrocyte membranome and its molecular composition. Objective: To develop a Triton X-114 based separation technique using nanoLC-MS/MS combined with shotgun proteomics to identify chondrocyte membrane proteins. Methods: Articular chondrocytes from equine metacarpophalangeal joints were separated into hydrophobic and hydrophilic fractions; trypsin-digested proteins were analysed by nanoLC-MS/MS. Results: A total of 315 proteins were identified. The phase extraction method yielded a high proportion of membrane proteins (56%) including CD276, S100-A6 and three VDAC isoforms. Conclusions: Defining the chondrocyte membranome is likely to reveal new biomarker targets for conventional and biological drug discovery.
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Publication Date: 2016-02-10 PubMed ID: 26864288PubMed Central: PMC4819840DOI: 10.3109/1354750X.2015.1130191Google Scholar: Lookup
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  • Journal Article
  • Research Support
  • Non-U.S. Gov't

Summary

This research summary has been generated with artificial intelligence and may contain errors and omissions. Refer to the original study to confirm details provided. Submit correction.

The study aims to identify proteins in the membranes of articular chondrocytes through a novel separation technique using Triton X-114. The findings could potentially reveal new biomarkers for drug discovery.

Objective of the Study

  • Given the paucity of knowledge concerning chondrocyte membranomes, the researchers set out to enhance understanding by identifying the membrane proteins specific to chondrocytes, essentially ‘defining the chondrocyte membranome’. The ultimate aim of this research is to discover new biomarker targets that can be exploited in drug discovery.

Methodology

  • The researchers isolated articular chondrocytes (the only cellular components of cartilage) from equine metacarpophalangeal joints. The isolation process involved parting the cells into hydrophobic (water-repellent) and hydrophilic (water-attracting) fractions.
  • The proteins in these fractions were digested with trypsin, an enzyme that breaks down proteins into peptides. This step was crucial for the nanoLC-MS/MS (nanoscale liquid chromatography-tandem mass spectrometry) analysis, as this technique can only analyze peptides and not entire proteins.
  • nanoLC-MS/MS in combination with shotgun proteomics was then employed to elucidate the identities of these membrane proteins. Shotgun proteomics is a large-scale technique that utilises mass spectrometry to identify proteins from a complex mixture.

Results

  • In total, the new technique identified 315 proteins. The assortment consisted of a high percentage of membrane proteins (56%), exemplifying the near-purification efficacy of the extraction technique.
  • The identified proteins included CD276 and S100-A6 proteins, as well as three isoforms of Voltage-Dependent Anion Channels (VDACs). Both CD276 and S100-A6 proteins are known to play crucial roles in cellular functioning and VDACs govern the entry and exit of mitochondrial molecules.

Conclusions and Implications

  • This study presents a novel technique for membrane protein extraction and identification, broadening our understanding of the chondrocyte membranome.
  • By identifying a high proportion of membrane proteins, the method could pave the way for characterizing these proteins, and could possibly reveal new biomarker targets for therapeutic interventions in diseases affecting the articular cartilage, like osteoarthritis.

Cite This Article

APA
Matta C, Zhang X, Liddell S, Smith JR, Mobasheri A. (2016). Label-free proteomic analysis of the hydrophobic membrane protein complement in articular chondrocytes: a technique for identification of membrane biomarkers. Biomarkers, 20(8), 572-589. https://doi.org/10.3109/1354750X.2015.1130191

Publication

Biomarkers : biochemical indicators of exposure, response, and susceptibility to chemicals
ISSN: 1366-5804
NlmUniqueID: 9606000
Country: England
Language: English
Volume: 20
Issue: 8
Pages: 572-589

Researcher Affiliations

Matta, Csaba
  • a Department of Veterinary Preclinical Sciences , School of Veterinary Medicine, Faculty of Health and Medical Sciences, University of Surrey , Guildford , Surrey , UK .
  • b Department of Anatomy , Histology and Embryology, Faculty of Medicine, University of Debrecen , Debrecen , Hungary .
Zhang, Xiaofei
  • c Proteomics Laboratory, School of Biosciences, University of Nottingham , Sutton Bonington , UK .
Liddell, Susan
  • c Proteomics Laboratory, School of Biosciences, University of Nottingham , Sutton Bonington , UK .
Smith, Julia R
  • d Bruker UK Limited , Coventry , UK .
Mobasheri, Ali
  • a Department of Veterinary Preclinical Sciences , School of Veterinary Medicine, Faculty of Health and Medical Sciences, University of Surrey , Guildford , Surrey , UK .
  • e Arthritis Research UK Centre for Sport, Exercise and Osteoarthritis, Arthritis Research UK Pain Centre, Medical Research Council and Arthritis Research UK Centre for Musculoskeletal Ageing Research, University of Nottingham, Queen's Medical Centre , Nottingham , UK , and.
  • f Center of Excellence in Genomic Medicine Research (CEGMR), King Fahd Medical Research Centre (KFMRC), Faculty of Applied Medical Sciences, King AbdulAziz University , Jeddah , Kingdom of Saudi Arabia.

MeSH Terms

  • Animals
  • B7 Antigens / metabolism
  • Biomarkers / metabolism
  • Cartilage, Articular / cytology
  • Cartilage, Articular / metabolism
  • Cells, Cultured
  • Chondrocytes / cytology
  • Chondrocytes / metabolism
  • Computational Biology
  • Detergents / chemistry
  • Horses
  • Hydrophobic and Hydrophilic Interactions
  • Liquid-Liquid Extraction
  • Membrane Proteins / chemistry
  • Membrane Proteins / metabolism
  • Metacarpophalangeal Joint / cytology
  • Metacarpophalangeal Joint / metabolism
  • Octoxynol
  • Polyethylene Glycols / chemistry
  • Proteomics / methods
  • S100 Proteins / metabolism
  • Tandem Mass Spectrometry
  • Voltage-Dependent Anion Channels / metabolism

Grant Funding

  • 20194 / Arthritis Research UK

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Citations

This article has been cited 4 times.
  1. Kovács P, Pushparaj PN, Takács R, Mobasheri A, Matta C. The clusterin connectome: Emerging players in chondrocyte biology and putative exploratory biomarkers of osteoarthritis.. Front Immunol 2023;14:1103097.
    doi: 10.3389/fimmu.2023.1103097pubmed: 37033956google scholar: lookup
  2. Jeremiasse B, Matta C, Fellows CR, Boocock DJ, Smith JR, Liddell S, Lafeber F, van Spil WE, Mobasheri A. Alterations in the chondrocyte surfaceome in response to pro-inflammatory cytokines.. BMC Mol Cell Biol 2020 Jun 26;21(1):47.
    doi: 10.1186/s12860-020-00288-9pubmed: 32586320google scholar: lookup
  3. Matta C, Boocock DJ, Fellows CR, Miosge N, Dixon JE, Liddell S, Smith J, Mobasheri A. Molecular phenotyping of the surfaceome of migratory chondroprogenitors and mesenchymal stem cells using biotinylation, glycocapture and quantitative LC-MS/MS proteomic analysis.. Sci Rep 2019 Jun 21;9(1):9018.
    doi: 10.1038/s41598-019-44957-ypubmed: 31227739google scholar: lookup
  4. Mobasheri A, Henrotin Y. Biomarkers of (osteo)arthritis.. Biomarkers 2015;20(8):513-8.
    doi: 10.3109/1354750X.2016.1140930pubmed: 26954784google scholar: lookup
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