Hofmann Andreas, Bausch-Fluck Damaris, Wollscheid Bernd
Institute of Molecular Systems Biology, Swiss Federal Institute of Technology (ETH) Zurich, Zurich, Switzerland.
Methods Mol Biol. 2013;951:33-43. doi: 10.1007/978-1-62703-146-2_3.
Cell surface glycoproteins represent important markers for the phenotyping of healthy and malignantly transformed cells. The mass spectrometry-based cell surface capturing (CSC) technology allows for extensive multiplexed identification and relative quantification of glycoproteins expressed on the cell surface at a given point in time. CSC technology is based on the mild oxidation of glycans from cell surface proteins on living cells. Oxidized glycans are tagged with a bifunctional linker molecule and glycopeptides are subsequently enriched by affinity chromatography. Here, we describe a step-by-step protocol of the CSC technology, which not only enables the identification of cell surface glycoproteins, but also the concurrent determination of protein N-glycosylation sites.
细胞表面糖蛋白是健康细胞和恶性转化细胞表型分析的重要标志物。基于质谱的细胞表面捕获（CSC）技术能够在给定时间点对细胞表面表达的糖蛋白进行广泛的多重鉴定和相对定量。CSC技术基于对活细胞表面蛋白聚糖的温和氧化。氧化后的聚糖用双功能连接分子进行标记，随后通过亲和色谱法富集糖肽。在此，我们描述了CSC技术的详细步骤方案，该方案不仅能够鉴定细胞表面糖蛋白，还能同时确定蛋白质的N-糖基化位点。
