Application of Fluorescently-Labeled Glycosphingolipids to Metabolic Profiling in Single Cells Using Capillary Electrophoresis

Abstract

Glycosphingolipids (GSLs) are important components expressed at the cell surface, where they play numerous roles in biological processes. The analysis of GSL metabolism is important but remains challenging in part due to the complexity of the components. To better understand the functions of GSLs on single cell scale we are developing an analytical method that uses fluorescently-tagged GSL analogs and capillary electrophoresis with laser induced fluorescence detection. We are applying this method to monitor glycolipid metabolism in single cells. In this article, we summarize our methodology, show examples of the chemoenzymatic synthesis of glycolipid analogs, and provide some experimental data in single cell analysis.

Synthetic Strategies for Modified Glycosphingolipids and Their Design as Probes

“…Complex gangliosides with fluorescent labels in the acyl chain have been prepared by first modifying the amine of lyso-LacCer or GM1 with a BODIPY or TMR fatty acid through NHS activation, followed by sialyltransferase, sialidase, or galactosidase reactions to generate the desired glycan. ,,,− These probes have been applied in metabolic cytometry experiments. , A convergent approach employing both synthetic and chemoenzymatic methods generated TMR-labeled gangliosides with a thioglycosidic linkage between galactose and glucose. Sph was glycosylated with a thiolactoside, after which the amine was acylated with a TMR label.…”

Analysis of the Cellular Dynamics of Fluorescently Tagged Glycosphingolipids by Using a Nanoliquid Chromatography–Tandem Mass Spectrometry Platform

“…Understanding the transformation process during glycan synthesis and degradation requires quantitative analysis of the individual molecules being converted; thus, the analysis should deal with spatiotemporal resolution − and molecular structure. − We planned to investigate the spatiotemporal transformation of glycosphingolipids occurring inside the cell, which can be achieved by conducting a time-resolved analysis of the localization of probe molecules together with analysis of their structure and quantity. For this purpose, it is logical to use a fluorescently tagged molecular probe, which is advantageous for live cell imaging of cellular events by using a microscope as well as for pulse-chase investigation of such molecules, − which are easily distinguishable from endogeneous GSLs. The choice and use of the fluorescent tag is critical but has to be chosen carefully because, in general, about half of the ceramide portion is replaced by the fluorescent moiety. , Such fluorescent molecules are still considered excellent probes for investigating cellular events mimicking the natural GSLs.…”

“…The choice and use of the fluorescent tag is critical but has to be chosen carefully because, in general, about half of the ceramide portion is replaced by the fluorescent moiety. , Such fluorescent molecules are still considered excellent probes for investigating cellular events mimicking the natural GSLs. Indeed, (4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoyl; BODIPY FL C5) lactosyl sphingosine (Lac-Sph) ( 1 ) has been used in investigating the biotransformation of glycan. − Furthermore, the laser-induced fluorescence (LIF) detection coupled with capillary electrophoresis made single cell analysis of 1 possible utilizing a series of chemically synthesized standard compounds. − However, because of the high sensitivity of LIF detection, structural analysis of unknown molecular species might be problematic. To avoid the potential problem taking advantage of mass spectrometry (MS), the use of several thousands of cells might be more convincing.…”