title: Separation of fluorescently labeled phosphoinositides and sphingolipids by capillary electrophoresis.
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 NaH(2)PO(4) and 1-propanol was optimized to separate two sphingolipids and multiple phosphoinositides by CE. The NaH(2)PO(4) 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 2×10(5) 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.
authors.affiliation: Department of Chemistry, University of North Carolina, Chapel Hill, NC 27599, United States.
authors.forename: Kelong
authors.initials: K
authors.lastname: Wang
authors.forename: Dechen
authors.initials: D
authors.lastname: Jiang
authors.forename: Christopher E
authors.initials: CE
authors.lastname: Sims
authors.forename: Nancy L
authors.initials: NL
authors.lastname: Allbritton
mesh_terms: D000433:1-Propanol / Q000737:chemistry; D001896:Boron Compounds / Q000737:chemistry; D045744:Cell Line, Tumor; D019075:Electrophoresis, Capillary / Q000379:methods*; D019793:Fluorescein / Q000737:chemistry; D005456:Fluorescent Dyes / Q000737:chemistry*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D057230:Limit of Detection; D010710:Phosphates / Q000737:chemistry; D010716:Phosphatidylinositols / Q000737:chemistry / Q000302:isolation & purification*; D015203:Reproducibility of Results; D013107:Sphingolipids / Q000737:chemistry / Q000302:isolation & purification*
chemical_list: C095489:4,4-difluoro-4-bora-3a,4a-diaza-s-indacene; D001896:Boron Compounds; D005456:Fluorescent Dyes; D010710:Phosphates; D010716:Phosphatidylinositols; D013107:Sphingolipids; D000433:1-Propanol; C018279:sodium phosphate; D019793:Fluorescein