Figure 2. LC-MS/MS based assay reveals that GST-PIP4Kα is highly specific for PI5P and has lower affinity for PI3P.

(A) TLC shows radioactive products formed from increasing concentrations of diC16-PI5P or diC16-PI3P (Echelon) upon kinase reaction with GST-PIP4Kα (see ‘Materials and methods’ section for details). Both assays have a ‘no enzyme control’ for 12 picomoles of lipid. PI(4,5)P₂ or PI(3,4)P₂ spots migrate with different R_f and are labelled on the TLC along with the origin. (B) Pixel intensity of the TLC image from (A) has been quantified and plotted for both PI5P and PI3P. Equations for PI5P: y = 250.32x + 53.897; PI3P: y = 12.771x + 9.5691. (C) Intensity response (cps) of respective products of PI3P and PI5P, using different substrate amounts from a ¹⁸O ATP-based kinase assay experiment analyzed by LC-MS/MS using d5-diC16-PI5P or 17:0 20:4 PI3P as indicated in the X-axis. Equations for PI5P: y = 575245x − 841692; PI3P: y = 25265x − 39253. (D) Response ratio of PI5P vs PI3P at different molar ratios of PI5P to PI3P. Y-axis represents response ratio of either d5-¹⁸O-PIP₂ to d5-PI5P (PI5P) or 17:0 20:4 ¹⁸O-PIP₂ to 17:0 20:4 PI3P and X-axis represents molar ratio of PI5P:PI3P. (E) TLC shows single radioactive spot corresponding to PI(4,5)P₂ from larvae of WT or dPIP4K²⁹ (mutant of dPIP4K).