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This protocol outlines a comprehensive approach for characterizing the cell surface subproteome of chondroprogenitor cells, based on aminooxy biotinylation followed by mass spectrometry analysis. The first step involves the selective labeling of cell surface proteins with aminooxy biotin on living chondroprogenitor cells, ensuring the specific tagging of glycoproteins on the outer membrane. Subsequently, glycocapture technique is employed to enrich the glycosylated fraction of the cell surface proteins. Following multiple wash steps to reduce contamination with detergents and nonsurface proteins, shotgun mass spectrometry is applied for the quantitative and qualitative analysis of the enriched subproteome, allowing for the identification and characterization of surface proteins. The integration of these techniques offers a comprehensive and sensitive method for profiling the cell surface proteome during chondrogenesis, enabling a deeper understanding of the molecular composition of chondroprogenitor cells. This protocol holds promise for advancing our knowledge of chondrogenesis and may contribute to the identification of potential therapeutic targets for cartilage-related disorders.

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Aminooxy Biotin-Based Characterization of the Surfaceome of Chondrogenic Cells

  • 实验方案

  • 发表日期：

    2025年

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  • Patrik Kovács,
  • David J. Boocock,
  • Clare Coveney,
  • Ali Mobasheri,
  • Csaba Matta

摘要

This protocol outlines a comprehensive approach for characterizing the cell surface subproteome of chondroprogenitor cells, based on aminooxy biotinylation followed by mass spectrometry analysis. The first step involves the selective labeling of cell surface proteins with aminooxy biotin on living chondroprogenitor cells, ensuring the specific tagging of glycoproteins on the outer membrane. Subsequently, glycocapture technique is employed to enrich the glycosylated fraction of the cell surface proteins. Following multiple wash steps to reduce contamination with detergents and nonsurface proteins, shotgun mass spectrometry is applied for the quantitative and qualitative analysis of the enriched subproteome, allowing for the identification and characterization of surface proteins. The integration of these techniques offers a comprehensive and sensitive method for profiling the cell surface proteome during chondrogenesis, enabling a deeper understanding of the molecular composition of chondroprogenitor cells. This protocol holds promise for advancing our knowledge of chondrogenesis and may contribute to the identification of potential therapeutic targets for cartilage-related disorders.