Hubei Key Laboratory of Diabetes and Angiopathy, Xianning Medical Colloge, Hubei University of Science and Technology, Xianning 437100, China.
School of Pharmacy, Xianning Medical Colloge, Hubei University of Science and Technology, Xianning 437100, China.
Acta Biochim Biophys Sin (Shanghai). 2022 Dec 25;54(12):1841-1853. doi: 10.3724/abbs.2022184.
TurboID, a proximity labelling method based on mutant biotin ligase, is an efficient new technique for recognizing protein-protein interactions and has been successfully applied to living cells. Sialic acid is typically the terminal monosaccharide attached to many glycoproteins and plays many important roles in many biological processes. However, the lack of enrichment methods for terminal sialic acid glycosylation hinders the identification and analysis of this glycosylation. Here, we introduce TurboID to identify terminal sialic acid glycosylation in living cells. CBM, the carbohydrate-binding domain of sialidase from , is fused with TurboID and overexpressed in HeLa cells. After streptavidin-based purification and detection by mass spectrometry, 31 terminal sialic acid N-glycosylated sites and 1359 putative terminal sialic acid glycosylated proteins are identified, many of which are located in the cytoplasm and nucleus.
TurboID 是一种基于突变生物素连接酶的邻近标记方法，是一种识别蛋白质-蛋白质相互作用的有效新技术，已成功应用于活细胞。唾液酸通常是连接到许多糖蛋白上的末端单糖，在许多生物过程中发挥着许多重要作用。然而，缺乏末端唾液酸糖基化的富集方法，阻碍了对这种糖基化的鉴定和分析。在这里，我们引入 TurboID 来鉴定活细胞中的末端唾液酸糖基化。CBM，神经氨酸酶的碳水化合物结合结构域来自 ，与 TurboID 融合并在 HeLa 细胞中过表达。经过基于链霉亲和素的纯化和质谱检测，鉴定出 31 个末端唾液酸 N-糖基化位点和 1359 个推定的末端唾液酸糖基化蛋白，其中许多位于细胞质和细胞核中。
