Hajdú Tibor, Nguyen Minh Ngoc, Zsigrai Emese, Coveney Clare, Boocock David J, Matta Csaba
Department of Anatomy, Histology and Embryology, Faculty of Medicine, University of Debrecen, Debrecen, Hungary.
Department of Pediatrics, Faculty of Medicine, University of Debrecen, Debrecen, Hungary.
Methods Mol Biol. 2025;2908:81-98. doi: 10.1007/978-1-0716-4434-8_6.
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.
黑素细胞在皮肤色素沉着以及抵御紫外线辐射方面发挥着关键作用。了解调控黑素细胞功能的分子机制对于阐明皮肤色素沉着紊乱和黑色素瘤进展至关重要。本方案详细介绍了一种全面的方法，用于利用氨氧基生物素化随后进行质谱分析来表征黑素细胞和黑色素瘤细胞系的细胞表面亚蛋白质组。第一步是在活的黑素细胞和黑色素瘤细胞系上用氨氧基生物素选择性标记细胞表面蛋白，以特异性标记糖蛋白。然后将标记的蛋白捕获在中性抗生物素蛋白磁珠上。经过多次洗涤步骤以尽量减少非细胞表面蛋白的污染后，采用质谱对富集的亚蛋白质组进行定量和定性分析，从而有助于鉴定和表征表面蛋白。这是一种在体外分析黑素细胞和黑色素瘤细胞系中细胞表面亚蛋白质组的灵敏方法，能更深入地了解它们的分子组成。该方案有望增进我们对黑色素瘤的理解，并可能有助于识别黑色素瘤治疗的潜在治疗靶点。
