Metabolic Cytometry: Capillary Electrophoresis with Two-Color Fluorescence Detection for the Simultaneous Study of Two Glycosphingolipid Metabolic Pathways in Single Primary Neurons
Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, Indiana 46556, United States.Analytical Chemistry (Impact Factor: 5.64). 03/2012; 84(6):2799-804. DOI: 10.1021/ac2031892
Metabolic cytometry is a form of chemical cytometry wherein metabolic cascades are monitored in single cells. We report the first example of metabolic cytometry where two different metabolic pathways are simultaneously monitored. Glycolipid catabolism in primary rat cerebella neurons was probed by incubation with tetramethylrhodamine-labeled GM1 (GM1-TMR). Simultaneously, both catabolism and anabolism were probed by coincubation with BODIPY-FL labeled LacCer (LacCer-BODIPY-FL). In a metabolic cytometry experiment, single cells were incubated with substrate, washed, aspirated into a capillary, and lysed. The components were separated by capillary electrophoresis equipped with a two-spectral channel laser-induced fluorescence detector. One channel monitored fluorescence generated by the metabolic products produced from GM1-TMR and the other monitored the metabolic products produced from LacCer-BODIPY-FL. The metabolic products were identified by comparison with the mobility of a set of standards. The detection system produced at least 6 orders of magnitude dynamic range in each spectral channel with negligible spectral crosstalk. Detection limits were 1 zmol for BODIPY-FL and 500 ymol for tetramethylrhodamine standard solutions.
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ABSTRACT: Phosphoinositides (PIs) and sphingolipids regulate many aspects of cell behavior and are often involved in disease processes such as oncogenesis. Capillary electrophoresis with laser induced fluorescence detection (CE-LIF) is emerging as an important tool for enzymatic assays of the metabolism of these lipids, particularly in cell-based formats. Previous separations of phosphoinositide lipids by CE required a complex buffer with polymer additives which had the disadvantages of high cost and/or short shelf life. Further a simultaneous separation of these classes of lipids has not been demonstrated in a robust buffer system. In the current work, a simple separation buffer based on NaH2PO4 and 1-propanol was optimized to separate two sphingolipids and multiple phosphoinositides by CE. The NaH2PO4 concentration, pH, 1-propanol fraction, and a surfactant additive to the buffer were individually optimized to achieve simultaneous separation of the sphingolipids and phosphoinositides. Fluorescein-labeled sphingosine (SFL) and sphingosine 1-phosphate (S1PFL), fluorescein-labeled phosphatidyl-inositol 4,5-bisphosphate (PIP2) and phosphatidyl-inositol 3,4,5-trisphosphate (PIP3), and bodipy-fluorescein (BFL)-labeled PIP2 and PIP3 were separated pairwise and in combination to demonstrate the generalizability of the method. Theoretical plate numbers achieved were as high as 2 × 105 in separating fluorophore-labeled PIP2 and PIP3. Detection limits for the 6 analytes were in the range of 10−18–10−20 mol. The method also showed high reproducibility, as the relative standard deviation of the normalized migration time for each analyte in the simultaneous separation of all 6 compounds was less than 1%. The separation of a mixture composed of diacylglycerol (DAG) and multiple phosphoinositides was also demonstrated. As a final test, fluorescent lipid metabolites formed within cells loaded with BFLPIP2 were separated from a cell lysate as well as a single cell. This simple and robust separation method for SFL and S1PFL and various metabolites of phosphoinositide-related signal transduction is expected to enable improved enzymatic assays for biological and clinical applications.Journal of Chromatography B 10/2012; 907:79-86. DOI:10.1016/j.jchromb.2012.09.003 · 2.73 Impact Factor
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ABSTRACT: Clonal microbial cells do not behave in an identical manner and form subpopulations during cultivation. Besides varying micro-environmental conditions, cell inherent features like cell cycle dependent localization and concentration of regulatory proteins as well as epigenetic properties are well accepted mechanisms creating cell heterogeneity. Another suspected reason is molecular noise on the transcriptional and translational level. A promising tool to unravel reasons for cell heterogeneity is the combination of cell sorting and subpopulation proteomics. This review summarizes recent developments in prokaryotic single-cell analytics and provides a workflow for selection of single cells, low cell number mass spectrometry, and proteomics evaluation. This approach is useful for understanding the dependency of individual cell decisions on inherent protein profiles.Current Opinion in Biotechnology 11/2012; 24(1). DOI:10.1016/j.copbio.2012.10.017 · 7.12 Impact Factor
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ABSTRACT: A capillary electrophoresis system with an ultrasensitive three-color laser-induced fluorescence detector was constructed for the simultaneous measurement of glycosphingolipids conjugated with a family of BODIPY fluorophores. The compounds were separated by capillary electrophoresis and detected by laser-induced fluorescence excited within a sheath-flow cuvette. Diode-pumped solid-state lasers operating at 473 nm and 532 nm, and a diode laser operating at 633 nm were used to excite glycosphingolipids tagged with BODIPY-FL, BODIPY-TMR, and BODIPY-650/665 fluorophores. Detection limits were 34 ± 1 molecules, 67 ± 7 molecules, and 36 ± 13 molecules of BODIPY-FL, BODIPY-TMR, and BODIPY-650/665 labeled glycosphingolipids. Separation efficiencies were typically one million theoretical plates. To test the ability of the system to analyze cellular contents in an in vitro biological model, differentiated PC12 cells were co-incubated with BODIPY-FL, BODIPY-TMR, and BODIPY-650/665 labeled lactosylceramide substrates. Cells were homogenized. The metabolic products originating from the glycosphingolipid substrates were simultaneously analyzed using the system.The Analyst 11/2012; 138(1). DOI:10.1039/c2an36286d · 4.11 Impact Factor
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