Whitmore Colin D, Olsson Ulf, Larsson E Andreas, Hindsgaul Ole, Palcic Monica M, Dovichi Norman J
Department of Chemistry, University of Washington, Seattle, WA, USA.
Electrophoresis. 2007 Aug;28(17):3100-4. doi: 10.1002/elps.200700202.
We report an ultrasensitive method for the analysis of glycosphingolipid catabolism. The substrate G(M1) and the set of seven metabolites into which it can be degraded (G(A1), G(M2), G(A2), G(M3), LacCer, GlcCer, and Cer) were labeled with the highly fluorescent dye tetramethylrhodamine. CE with LIF detection was used to assay these compounds with 150 +/- 80 yoctomole mass (1 ymol = 10(-24) mol = 0.6 copies) detection limits and 5 +/- 3 pM concentration detection limits. An alignment algorithm based on migration of two components was employed to correct for drift in the separation. The within-day and between-day precision in peak height was 20%, in peak width 15%, and in adjusted migration time 0.03%. After normalization to total sample injected, the RSD in peak height reduced to 2-6%, which approaches the limit set by molecular shot noise in the number of molecules taken for analysis. PC12 cells were incubated with the labeled G(M1). Fluorescent microscopy demonstrated uptake by the cells. CE was used to separate a cellular homogenate prepared from these cells. A set of peaks was observed, which were tentatively identified based on comigration with the standards. Roughly 120 pL of homogenate was injected, which contained a total of 150 zmol of labeled substrate and products. Metabolite that preserves the fluorescent label can be detected at the yoctomole level, which should allow characterization of this metabolic pathway in single cells.
我们报告了一种用于分析糖鞘脂分解代谢的超灵敏方法。底物G(M1)及其可降解生成的七种代谢产物（G(A1)、G(M2)、G(A2)、G(M3)、乳糖神经酰胺、葡萄糖神经酰胺和神经酰胺）用高荧光染料四甲基罗丹明进行标记。采用毛细管电泳结合激光诱导荧光检测法对这些化合物进行测定，其质量检测限为150±80 yoctomole（1 ymol = 10⁻²⁴ mol = 0.6个拷贝），浓度检测限为5±3 pM。采用基于两种组分迁移的比对算法来校正分离过程中的漂移。日内和日间峰高的精密度为20%，峰宽为15%，调整迁移时间为0.03%。在对进样的总样品进行归一化处理后，峰高的相对标准偏差降至2 - 6%，这接近分析所用分子数中分子散粒噪声所设定的极限。将PC12细胞与标记的G(M1)一起孵育。荧光显微镜显示细胞摄取了该物质。采用毛细管电泳分离由这些细胞制备的细胞匀浆。观察到一组峰，根据与标准品的共迁移情况初步进行了鉴定。大约进样120 pL的匀浆，其中总共含有150 zmol的标记底物和产物。能够在yoctomole水平检测到保留荧光标记的代谢产物，这应该有助于在单细胞中表征这种代谢途径。
