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Melanocytes play a crucial role in skin pigmentation and protection against UV radiation. Understanding the molecular mechanisms regulating melanocyte function is essential for elucidating skin pigmentation disorders and melanoma progression. This protocol details a comprehensive methodology for characterizing the cell surface subproteome of melanocytes and melanoma cell lines, utilizing aminooxy biotinylation followed by mass spectrometry analysis. The initial step involves selectively labeling cell surface proteins with aminooxy biotin on live melanocytes and melanoma cell lines to specifically tag glycoproteins. Labeled proteins are then captured on NeutrAvidin beads. Following multiple wash steps to minimize contamination with non-cell surface proteins, mass spectrometry is employed for quantitative and qualitative analysis of the enriched subproteome, facilitating the identification and characterization of surface proteins. This is a sensitive method for profiling the cell surface subproteome in melanocytes and melanoma cell lines in vitro, offering deeper insights into their molecular composition. This protocol holds promise for advancing our understanding of melanoma and may aid in identifying potential therapeutic targets for melanoma treatment.

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Isolation of the Surfaceome from Primary Human Epidermal Melanocytes Using Glycocapture and Shotgun Proteomics

  • 实验方案

  • 发表日期：

    2025年

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  • Tibor Hajdú,
  • Minh Ngoc Nguyen,
  • Emese Zsigrai,
  • Clare Coveney,
  • David J. Boocock,
  • Csaba Matta

摘要

Melanocytes play a crucial role in skin pigmentation and protection against UV radiation. Understanding the molecular mechanisms regulating melanocyte function is essential for elucidating skin pigmentation disorders and melanoma progression. This protocol details a comprehensive methodology for characterizing the cell surface subproteome of melanocytes and melanoma cell lines, utilizing aminooxy biotinylation followed by mass spectrometry analysis. The initial step involves selectively labeling cell surface proteins with aminooxy biotin on live melanocytes and melanoma cell lines to specifically tag glycoproteins. Labeled proteins are then captured on NeutrAvidin beads. Following multiple wash steps to minimize contamination with non-cell surface proteins, mass spectrometry is employed for quantitative and qualitative analysis of the enriched subproteome, facilitating the identification and characterization of surface proteins. This is a sensitive method for profiling the cell surface subproteome in melanocytes and melanoma cell lines in vitro, offering deeper insights into their molecular composition. This protocol holds promise for advancing our understanding of melanoma and may aid in identifying potential therapeutic targets for melanoma treatment.