Effective protein extraction combined with data independent acquisition analysis reveals a comprehensive and quantifiable insight into the proteomes of articular cartilage and subchondral bone

Louise Bundgaard*, Emma Åhrman, Johan Malmström, Ulrich auf dem Keller, Marie Walters, Stine Jacobsen

*Corresponding author for this work

Research output: Contribution to journalJournal articleResearchpeer-review

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Abstract

Objective
The objectives of this study was to establish a sensitive and reproducible method to map the cartilage and subchondral bone proteomes in quantitative terms, and mine the proteomes for proteins of particular interest in the pathogenesis of osteoarthritis (OA). The horse was used as a model animal.

Design
Protein was extracted from articular cartilage and subchondral bone samples from three horses in triplicate by pressure cycling technology or ultrasonication. Digested proteins were analysed by data independent acquisition based mass spectrometry. Data was processed using a pre-established spectral library as reference database (FDR 1%).

Results
We identified to our knowledge the hitherto most comprehensive quantitative cartilage (1758 proteins) and subchondral bone (1482 proteins) proteomes in all species presented to date. Both extraction methods were sensitive and reproducible and the high consistency of the identified proteomes (>97% overlap) indicated that both methods preserved the diversity among the extracted proteins. Proteome mining revealed a substantial number of quantifiable cartilage and bone matrix proteins and proteins involved in osteogenesis and bone remodeling, including ACAN, BGN, PRELP, FMOD, COMP, ACP5, BMP3, BMP6, BGLAP, TGFB1, IGF1, ALP, MMP3, and collagens. A number of proteins, including COMP and TNN, were identified in different protein isoforms with potential unique biological roles.

Conclusion
We have successfully developed two sensitive and reproducible non-species specific workflows enabling a comprehensive quantitative insight into the proteomes of cartilage and subchondral bone. This facilitates the prospect of investigating the molecular events at the osteochondral unit in the pathogenesis of OA in future projects.
Original languageEnglish
JournalOsteoarthritis and Cartilage
Volume30
Issue number1
Pages (from-to)137-146
Number of pages10
ISSN1063-4584
DOIs
Publication statusPublished - 2022

Keywords

  • Cartilage
  • Subchondral bone
  • Proteome
  • Osteoarthritis
  • Data Independent Acquisition
  • Horse

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