Revision as of 10:28, 10 October 2010

acidic phospholipids (polyphosphoinositides)

Cultured cells (3 to 6 × 106 per 100 mm plate) were scraped with 2 mL of ice-cold methanol, 50 pmol of PIP1(16:0/16:0), PIP2(16:0/16:0), and PIP3(16:0/16:0) were added as internal standards and 2 μL of 1 nmol/μL PIP2(8:0/8:0) was added as an adsorption protectant.
2mL of 1 N HCl, 0.15 mL of 2 M NaCl, and 2 mL of chloroform were added to suspension, followed by vigorous shaking for 3 min while cooling with ice.
Samples were centrifuged at 1000g for 5 min, and the lower layer was collected.
After addition of 1.5 mL of methanol, each sample was applied to a DEAE-cellulose (Wako Pure Chemicals) column.
Column-bound lipids were washed with chloroform (1 mL), chloroform/methanol (1:1) (3 × 1 mL), and chloroform/methanol/28% aqueous ammonia/acetic acid (200:100:3:0.9) (3 × 1 mL) in series.
Highly acidic lipids, including PIPs, LPA, and LPS were eluted with chloroform/methanol/HCl (6:6:1) (3 × 1 mL).
After addition of 1.5 mL of water and 0.113 mL of 2 M NaCl, the solution was vigorously shaken and centrifuged to collect the lower layer.
After addition of 2 μL of 1 nmol/μL PIP2(8:0/8:0), each sample was dried under nitrogen gas.
The sample was redissolved in 0.05 mL of methanol/70% ethylamine (100:0.065), followed by addition of 0.03 mL of 1 M ammonium bicarbonate containing 0.2 mM phosphoric acid.

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