Metal cations for the determination of fluorescent phosphoinositides by capillary electrophoresis

Abstract

Phosphatidylinositol (PI) and its phosphorylated derivatives known as phosphoinositides (PIPs), are essential regulators of cell signaling and membrane trafficking, cytoskeletal dynamics, and nuclear functions. Disruption of PI metabolism is associated with disorders such as immune dysfunction, cardiovascular disease, and cancer; therefore, there is currently great interest in studying PIPs and their metabolic enzymes. Here, we describe a method for the separation of fluorescent PI and its seven fluorescent phosphorylated derivatives by CE-LIF. The CE method utilizes a Tris buffer and sodium deoxycholate in the presence of 30% 1-propanol and 5% of a dynamic coating reagent, EOTrol low reverse (EOTrol LR). It is simple, fast, highly sensitive, and it offers LODs in the order of 1.5 amol. The effect of cations such as lithium, sodium, potassium, cesium, barium, manganese, zinc, magnesium, calcium, spermine, and gentamicin were evaluated. Calcium and magnesium provided the best selectivity and resolution for the separation of the analytes while magnesium offered the best data reproducibility. The developed CE method would be useful in the studies of enzymatic activity in the PI and PIPs metabolic pathways using CE-based in vitro and CE cell-based assays, and/or for drug screening.

Capillary electrophoresis characterization of molecularly imprinted polymer particles in fast binding with 17β‐estradiol

“…As claimed by the manufacturer, their effects on EOF are nearly independent of pH and buffer types. Addition of these polymers at a very low concentration to the running buffer (or BGE) can increase, suppress, or even reverse the EOF at any buffer pH between 3 and 11 35. To minimize the impact of EOTrol™ absorbance (at ∼210 nm) on the UV detection of MIP and NIP particles (214 nm), a low concentration of 0.005% EOTrol™ LN was used in 50 mM borate buffer (pH=8.5).…”

Single-cell analysis of phosphoinositide 3-kinase and phosphatase and tensin homolog activation

“…Separation of Bodipy Fl PIP 2 and Bodipy Fl PIP 3 was accomplished as previously reported 60, 61. The current work sought to apply this separation scheme to single-cell analyses of PIP 2 and PIP 3 metabolism; therefore, it was necessary to extend the earlier work to establish that adequate sensitivity could be attained to accomplish this goal.…”

“…Separation and detection limits of Bodipy Fl PIP 2 and Bodipy Fl PIP 3 by CE Separation of Bodipy Fl PIP 2 and Bodipy Fl PIP 3 was accomplished as previously reported. 60,61 The current work sought to apply this separation scheme to singlecell analyses of PIP 2 and PIP 3 metabolism; therefore, it was necessary to extend the earlier work to establish that adequate sensitivity could be attained to accomplish this goal. Due to the biochemical nature of the enzyme measurements, it was desired to introduce the fluorescent substrate at the lowest possible cellular concentration; otherwise, excessive amounts of the exogenous lipid could serve to overwhelm the cellular signaling networks with a substrate that might perturb cellular physiology.…”

Analysis of in vitro oxidized human LDL phospholipids by solid‐phase extraction and micellar electrokinetic capillary chromatography

“…Previously, CE has been used to study phospholipids. Nonaqueous CE has been used to analyze the major phospholipid classes, phosphatidylethanol and phosphoinositides (Raith et al ., ; Guo et al ., ; Nalesso et al ., ; Varga and Nilsson, ; Otieno et al ., ; Gao et al ., ). The CE method with indirect UV measurement has been used to separate lysophosphatidic acids, anionic phospholipids and phospholipid classes (Chen and Xu, ; Haddadian et al ., ; Lucangioli et al ., ; Sedláková et al ., ; Gao et al ., ).…”