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Abstract：
Biotin labeling in combination with mass spectrometry has been widely applied in large-scale biological studies, such as determination of protein partners, protein subcellular localization, and protein post-translational modifications. Previous studies have shown that immunoaffinity enrichment is a better method than streptavidin/avidin purification for site-specific studies of biotinylated molecules. In this study, we made a crucial improvement to the elution phase of the immunoaffinity enrichment step for biotinylated peptides, which involves the addition of a highly organic solvent, and developed a monoclonal anti-biotin antibody that improved the identification number for biotinylated peptides. We then demonstrated its application in the characterization of protein interaction sites for the β2 adrenergic receptor (β2AR) by proximity labeling in living cells. Our research provides an improved and reproducible immunoaffinity enrichment method for site-specific biotin-related research.
摘要：
生物素标记与质谱联用已广泛应用于大规模生物学研究,例如确定蛋白质伴侣，蛋白质亚细胞定位，和蛋白质翻译后修饰。先前的研究表明，对于生物素化分子的位点特异性研究，免疫亲和富集是比链霉亲和素/抗生物素蛋白纯化更好的方法。在这项研究中,我们对生物素化肽的免疫亲和富集步骤的洗脱阶段进行了关键的改进，其中包括添加高度有机溶剂，并开发了一种单克隆抗生物素抗体，该抗体提高了生物素化肽的鉴定编号。然后，我们通过活细胞中的邻近标记证明了其在表征β2肾上腺素能受体（β2AR）的蛋白质相互作用位点中的应用。我们的研究为位点特异性生物素相关研究提供了一种改进的、可重复的免疫亲和富集方法。
