HPLC Analysis of Glycerophosphoinositols see Note

The deacylated lipids are resolved using anion-exchange chromatography with a Whatman Partisil 5 SAX (4.6 X 250 mm) or Partisil 10 SAX (4.6 X 250 mm) column fitted with a guard column (Phenomenex, Torrance, CA). A Beck-man System Gold chromatograph equipped with a UV detector and Beckman System Gold software or equivalent system should be used. All samples should be spiked with internal controls of AMP, ADP, and ATP in order to monitor the column performance. Typically, a portion of each sample to be loaded on the column (5 X 106 cpm or more) can be mixed with 40 nmol each of AMP, ADP, ATP and applied to the column. For reference, glycerophosphoinositol phosphate species [gPI(3)P and gPI(4)P] elute between AMP and ADP, glycerophosphoinositol bisphosphate species [gPI(3,4)P2 and gPI(3,5)P2] and gPI(4,5)P2] elute between ADP and ATP, and gPI(3,4,5)P3 elutes just after ATP. Phosphoinositides can be resolved on the anion-exchange column with the following mobile phase: 5 mL of isocratic 10 mM ammonium phosphate (pH 3.8), 60 mL of a linear gradient (10-800 mM), a 10-mL gradient from 800 mM to 1000 mM, then 10 mL of 1000 mM of ammonium phosphate (pH 3.8) at a flow rate of 1 mL/min. Fractions should be collected every 20 s, mixed with 2-3 mL EcoLume (ICN), and counted in a liquid scintillation counter (Beckman LS 5801) (see Note 3).

Fig. 2. HPLC analysis of glycerophosphoinositols from PDGF-stimulated 3T3-L1 cells. 5 X 106 cpm of sample was injected onto a Partisil 10 SAX column and samples resolved using gradient described in the text. Fractions were collected and counted in a scintillation counter and counts in each fraction were plotted. gPI(3)P is glycerophos-phoinositol 3-phosphate, gPI(4)P is glycerophosphoinositol 4-phosphate, and the other glycerophosphoinositol species are likewise designated.

Fig. 2. HPLC analysis of glycerophosphoinositols from PDGF-stimulated 3T3-L1 cells. 5 X 106 cpm of sample was injected onto a Partisil 10 SAX column and samples resolved using gradient described in the text. Fractions were collected and counted in a scintillation counter and counts in each fraction were plotted. gPI(3)P is glycerophos-phoinositol 3-phosphate, gPI(4)P is glycerophosphoinositol 4-phosphate, and the other glycerophosphoinositol species are likewise designated.

Figure 2 illustrates a radioactivity profile generated from 3T3-L1 preadi-pocytes stimulated with PDGF. The profile displays all of the glycerophospho-inositols except gPI(5)P and provides a relative measure of the levels of phosphoinositides when compared to those from unstimulated cells. In addition, this profile illustrates the consistent order of elution of the phosphoinositides [gPI(5)P elutes just after gPI(4)P].

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