Over 80% of the conjugated saccharides in the central nervous system are in the form of lipids.1
Glycosphingolipids (GSLs) are ubiquitous on neuronal cell surfaces and are made up of a nonpolar tail consisting of a fatty acid and sphingosine with a polar sugar headgroup.2
The number of substitutions and saccharide composition varies, giving rise to a complex suite of GSLs that mediate a variety of functions including cellular differentiation, intracellular signalling, pathogen binding, and oncogenesis.3–6
Defects in GSL metabolism result in several debilitating disorders including Tay-Sachs disease and Sandhoff’s disease.7
Sensitive high-resolution measurement of GSLs presents a difficult analytical challenge. High performance thin layer chromatography is the traditional approach to quantify GSLs, but suffers from poor sensitivity.8
Gas chromatography and high performance liquid chromatography have been used to measure GSLs, but ideal separation conditions confound mass spectrometric detection.9–11
Our group pioneered the use of capillary electrophoresis with laser-induced fluorescence detection to monitor GSL metabolism in cellular homogenates and single cells.12–18
GSLs are chemoenzymatically prepared to include a fluorophore attached to the sphingosine tail.19, 20
Cells are incubated with these fluorescently-labeled GSLs and various metabolic products due to the addition (anabolism) or removal (catabolism) of saccharides from the sugar headgroup are detected.
We recently reported the development and characterization of a capillary electrophoresis system with two-color laser-induced fluorescence detection used to monitor GSL metabolism.13
Together, two diode-pumped solid-state lasers, one operating in the blue at 473 nm and the other operating in the green at 532 nm, were used to excite fluorescence from BODIPY-FL- and tetramethylrhodamine-labeled GSLs, respectively. Here, we report a three-color system with the addition of a 633 nm red diode laser.
We have observed that tetramethylrhodamine-labeled GSLs exclusively undergo catabolism, whereas BODIPY-FL GSLs undergo both catabolism and anabolism.13
Fluorescence microscopy revealed that compounds labeled with the two fluorophores had quite distinct cellular distribution, reflecting differences in the uptake and transport properties of these compounds. To further study the metabolic behaviour of GSLs labeled with a range of boron dipyrromethene (BODIPY) fluorophores, we chemoenzymatically prepared GSLs tagged with fluorophores within the BODIPY family: BODIPY-FL (λex
505 nm/513 nm), BODIPY-TMR (λex
542 nm /574 nm), and BODIPY-650/665 ((λex
646 nm /660 nm).21