Hamouda Houda, Kaup Matthias, Ullah Mujib, Berger Markus, Sandig Volker, Tauber Rudolf, Blanchard Véronique
Institute of Laboratory Medicine, Clinical Chemistry and Pathobiochemistry, Charité-Universitätsmedizin Berlin , Augustenburger Platz 1, 13353 Berlin, Germany.
J Proteome Res. 2014 Dec 5;13(12):6144-51. doi: 10.1021/pr5003005. Epub 2014 Nov 13.
Cell surfaces are covered with a dense carbohydrate layer referred to as the glycocalyx. Because different cell types express different glycan signatures, it is of paramount importance to have robust methods to analyze the glycome of living cells. To achieve this, a common procedure involves cell lysis and extraction of membrane (glyco)proteins and yields a major proportion of high-mannose N-glycans that most likely stem from intracellular proteins derived from the ER. Using HEK 293 cells as a model system, we developed a reproducible, sensitive, and fast method to profile surface N-glycosylation from living cells. We directly released glycopeptides from cell surfaces through tryptic digestion of freshly harvested and vital cells, thereby improving the detection and quantification of complex-type N-glycans by increasing their relative amount from 14 to 85%. It was also possible to detect 25 additional structures in HEK 293, 48 in AGE1.HN, 42 in CHO-K1, and 51 in Hep G2 cells. The additional signals provided deeper insight into cell-type-specific N-glycan features such as antennarity, fucosylation, and sialylation. Thus, this protocol, which can potentially be applied to any cells, will be useful in the fields of glycobiotechnology and biomarker discovery.
细胞表面覆盖着一层致密的碳水化合物层，称为糖萼。由于不同细胞类型表达不同的聚糖特征，因此拥有强大的方法来分析活细胞的糖组至关重要。为实现这一点，常见的步骤包括细胞裂解以及提取膜（糖）蛋白，并产生很大一部分高甘露糖型N-聚糖，这些聚糖很可能来源于内质网的细胞内蛋白质。我们以HEK 293细胞作为模型系统，开发了一种可重复、灵敏且快速的方法来分析活细胞表面的N-糖基化。我们通过对新鲜收获的活细胞进行胰蛋白酶消化，直接从细胞表面释放糖肽，从而将复合型N-聚糖的相对含量从14%提高到85%，改善了其检测和定量。在HEK 293细胞中还能够检测到另外25种结构，在AGE1.HN细胞中为48种，在CHO-K1细胞中为42种，在Hep G2细胞中为51种。这些额外的信号为细胞类型特异性的N-聚糖特征，如天线性、岩藻糖基化和唾液酸化，提供了更深入的见解。因此，该方案有可能应用于任何细胞，将在糖生物技术和生物标志物发现领域发挥作用。
