Mass Spectrometry Measurements of Inositol Lipids.

Mass spectrometry was used to measure inositol lipid levels essentially as described previously (77) using a QTRAP 4000 (AB Sciex) mass spectrometer and employing the lipid extraction and derivatization method described for cultured cells (cells isolated from tumors) and whole tissue (tumor tissue), with the modification that initial samples were probe sonicated for 5 seconds (using a microtip) prior to extraction and that final samples were dried in a speedvac concentrator rather than under N2. Measurements were conducted on 1 × 10⁶ isolated fibrosarcoma cells or 1 mg wet weight tumor tissue per sample. C16:0/C17:0 PI (100 ng) and PIP3 (10 ng) internal standards (ISDs) were added to each sample prior to extraction. Integrated area of lipid species peaks were corrected for recovery against ISD area (giving a response ratio for each lipid) and data expressed as C18:0 C20:4 PIP3 response ratio normalized to C18:0 C20:4 PI response ratio to account for cell input variation. Both endogenous PIP3 and PI were corrected to their own internal standard, and then the one measurement was divided by the other, to get the best estimate of true PIP3/PI.