Whitmore Colin D, Hindsgaul Ole, Palcic Monica M, Schnaar Ronald L, Dovichi Norman J
Department of Chemistry, University of Washington, Box 351700, Seattle, Washington 98195, USA.
Anal Chem. 2007 Jul 15;79(14):5139-42. doi: 10.1021/ac070716d. Epub 2007 Jun 14.
Glycosphingolipids are found on all vertebrate cells and constitute major cell surface determinants on all nerve cells, where they contribute to cellular diversity and function. We report a method for the analysis of glycosphingolipid metabolism in single cells. The ganglioside GM1 was tagged with the fluorescent dye tetramethylrhodamine. This labeled compound was taken up and metabolized by a culture of pituitary tumor (AtT-20) cells. After 50 h, the cells were formalin fixed. Cells were aspirated into a fused-silica capillary and lysed, and components were separated by capillary electrophoresis with a laser-induced fluorescence detector. All metabolic products that retained the fluorescent dye could be detected at the low-zeptomole level. A total of 54 AtT-20 cells were individually analyzed using this procedure. The electrophoretic profiles were remarkably reproducible, which facilitated identification of components based on the migration time of fluorescently labeled standards. Eleven components were detected, and the average peak height of these components spanned more than 2 orders of magnitude, so that trace metabolites can be detected in the presence of abundant components. The most highly abundant components generated 10% relative standard deviation in normalized abundance. The average cell took up roughly 2 amol (10(6) copies) of the labeled substrate. This method allows determination of cell-to-cell diversity and regulation of glycosphingolipid metabolism.
糖鞘脂存在于所有脊椎动物细胞中，是所有神经细胞主要的细胞表面决定簇，在神经细胞中它们对细胞多样性和功能有重要作用。我们报道了一种分析单细胞中糖鞘脂代谢的方法。神经节苷脂GM1用荧光染料四甲基罗丹明进行标记。这种标记化合物被垂体瘤（AtT-20）细胞培养物摄取并代谢。50小时后，将细胞用福尔马林固定。把细胞吸入熔融石英毛细管中进行裂解，然后用激光诱导荧光检测器通过毛细管电泳分离各组分。所有保留荧光染料的代谢产物都能在低zeptomole水平被检测到。使用该方法对总共54个AtT-20细胞进行了单独分析。电泳图谱具有显著的可重复性，这有助于根据荧光标记标准品的迁移时间来鉴定各组分。检测到了11种组分，这些组分的平均峰高跨越了2个多数量级，因此在存在大量组分的情况下也能检测到痕量代谢物。含量最高的组分在归一化丰度方面产生了10%的相对标准偏差。平均每个细胞摄取了大约2 amol（10⁶个拷贝）的标记底物。该方法能够确定细胞间糖鞘脂代谢的多样性和调控情况。
