Quantitative lipid profiling protocol

750,000 Mcf10a cells grown on a 35 mm dish were killed in 750 μL ice-cold 1 M HCL, then scraped and collected into an Eppendorf tube. Each sample was then split into three separate 2 mL polypropylene Eppendorf tubes; 250 μL for PI, PIP, PIP₂, PIP₃ measurement, 250 μL for PI(3,4)P₂/PI(4,5)P₂ measurement, and the remaining cells were kept for analysis by western blot. Cells were pelleted in a microfuge (15,000 x g, 10 min at 4°C), the supernatant removed and cell pellets either processed immediately or snap-frozen in liquid nitrogen and stored at −80°C for up to two weeks.
For human prostate and breast cancer cell lines, 250,000 cells were seeded into 35 mm dishes and grown in the medium optimal for each cell line for 32h. Cells were then starved for 16h by replacing the medium with starvation medium – a phenol red-free RPMI 1640 supplemented with 2mM glutamax. Following stimulation and / or inhibition with appropriate reagents, medium was removed by aspiration and cells killed with 750 μl ice-cold 1M HCL. Cells were then scraped, collected into Eppendorf tubes, pelleted, and snap-frozen, as described above.
920 μL of a solvent mixture containing 2:1:0.79 (v/v) MeOH:CHCl₃:H₂O_(acidic) was added to the cell pellets and the mixture vortexed thoroughly for 10 s. Relevant internal standards were then added:10 ng C17:0/C16:0-PIP₃, 100 ng C17:0/C16:0-PI, 250 ng d6-C18:0/C20:4-PI(4,5)P₂ for routine analysis of PI, PIP, PIP₂ and PIP₃; 50 ng C17:0/C20:4 PI, 50 ng d6- C18:0/C20:4-PI(3,4)P₂, 250 ng d6-C18:0/C20:4-PI(4,5)P₂ for routine analysis of PI, PI(3,4)P₂ and PI(4,5)P₂. Lipids were then extracted using an acidified Folch phase partition and derivatised with TMS-diazomethane (Clark et al., 2011 (link)).
Molecules derived from PI, PIP, PIP₂, and PIP₃ were measured by HPLC-MS (Kielkowska et al., 2014 (link)). Response ratios were calculated for the endogenous species of these lipids divided by their relevant C17:0/C16:0 internal standard. We routinely analyzed 5 molecular species of these lipids but present here data for the C38:4 species only, to align with data presented for the C38:4 species of PI(3,4)P₂ and PI(4,5)P₂ (see below). The C38:4 species of PIP₂ and PIP₃ represent approx. 10%–15% of the total species of these lipids in Mcf10a cells and all species behave very similarly upon stimulation with EGF (Anderson et al., 2016 (link)). In some experiments, absolute amounts of C38:4 PI(3,4,5)P₃ were generated by reference to standard curves previously generated for this molecular species (Kielkowska et al., 2014 (link)). Three technical replicates were routinely analyzed for each experiment and, unless stated otherwise, data are presented as means SD of three biological replicates.
Molecules derived from PI, PI(3,4)P₂ and PI(4,5)P₂ were analyzed by a new HPLC-MS method, see below. Response ratios were calculated for the endogenous C38:4 species of these lipids divided by their relevant d6-labeled internal standard. In some experiments, absolute amounts of these lipids were generated by reference to standard curves (Figure S1). Three technical replicates were routinely analyzed for each experiment and, unless stated otherwise, data are presented as means SD of three biological replicates.