Phosphoinositides are minor components of cell membranes, but play crucial roles in numerous signal transduction pathways. To obtain quantitative measures of phosphoinositides, sensitive, accurate, and comprehensive methods are needed. Here, we present a quantitative targeted ion chromatography−mass spectrometry-based workflow that separates phosphoinositides isomers and increases the quantitative accuracy of measured phosphoinositides. Besides testing different analytical characteristics such as extraction and separation efficiency, the reproducibility of the developed workflow was also investigated. The workflow was verified in resting and stimulated human platelets, fat cells and rat hippocampal brain tissue, where the LOD and LOQ for phosphoinositides were at 312.5 fmol and 625 fmol respectively. The robustness of the workflow is shown with different applications that confirms its suitability to analyze multiple low abundant phosphoinositides.

Lipid extraction. Acidified chloroform/methanol (CHCl3/MeOH) extraction was carried out following the protocol of Clark et al.16 For platelet samples, after addition of 242 μL CHCl3, 484 μL MeOH, 23.6 µL 1M HCl, 170 µL water and the internal standard (100 pmol of PtdIns(4,5)P2-FP) to the cell pellets containing 1x108 platelets, the mixture was allowed to stand at room temperature for 5 min with occasional vortexing. Next, 725 µL of CHCl3 and 170 µL 2M HCl was added to induce phase separation and the samples were centrifuged at 1 500 g for 5 min at room temperature (Eppendorf, Hamburg, Germany). This created a two-phase system with an upper aqueous layer and a protein interface. Then, the lower organic layer was transferred to another tube and dried under a continuous stream of nitrogen (1 L/min N2 at 25°C). For pre-adipocytes and rat hippocampal heavy membrane fraction, after the addition of 242 μL CHCl3, 484 μL MeOH, 25 µL 50 mM NaOH, 170 µL water and the internal standard (2 nmol of PI(4,5)P2-FP) to the cell pellets, the mixture was vortexed and sonicated until homogenization. Afterwards, 725 µL CHCl3 was added, and the samples were centrifuged at 1 500 g for 5 min at room temperature. The resulting lower phase containing neutral lipids was removed without disturbing the upper aqueous phase and protein interphase. Next, 170 µL 2M HCl, 333 µL MeOH and 667 µL CHCl3 was added to the remaining phase, and the mixture was allowed to stand at room temperature for 5 min with occasional vortexing. The samples were then centrifuged at 1 500 g for 5 min at room temperature. Next, the lower organic layer was transferred to another tube and dried under a continuous stream of nitrogen (1 L/min N2 at 25°C). The lipid extracts were then deacylated following the protocol of Jeschke et al.18 The dried lipid extracts were resuspended in 50 µL methylamine in methanol/water/1-butanol (46:43:11) and incubated at 53°C for 50 min in a thermomixer at 1 000 rpm (Thermomixer Comfort; Eppendorf, Hamburg, Germany). Then 25 µL cold IPA was added to the mixture, and the mixture was dried under a continuous stream of nitrogen to obtain dried lipid extracts (1 L/min N2 at 25°C). The dried and deacylated lipid extract was resuspended in 50 μL water and stored at -80°C prior to further analysis. Protein concentration determination. 1200 µL methanol was added to the remaining protein interphase and aqueous upper phase, and the mixture was incubated at -80°C for 3 h. Then the mixture was centrifuged at 19,000 g for 30 min at 4 °C, the supernatant removed, and the remaining protein pellet dried under fume hood. The dried protein was then dissolved in 1% SDS, 150 mM NaCl, 50 mM Tris (pH 7.8) for protein concentration determination using BCA assay. IC−MS/MS. IC-MS/MS was conducted using a Dionex ICS-5000 instrument (Thermo Fischer Scientific, Darmstadt, Germany) connected to a QTRAP 6500 instrument (AB Sciex, Darmstadt, Germany) that was equipped with an electrospray ion source (Turbo V ion source). Chromatographic separation was accomplished with a Dionex IonPac AS11-HC column (250 mm × 2 mm, 4 μm; Thermo Fischer Scientific) fitted with a guard column (50 mm × 2 mm, 4 μm; Thermo Fischer Scientific). A segmented linear gradient was used for separation of GroPInsP: Initial 15 mM potassium hydroxide (KOH), held at 15 mM KOH from 0.0 to 5.0 min, 15 to 25 mM KOH from 5.0 to 15.0 min, 50 to 65 mM KOH from 15.0 to 30.0 min, 100 mM KOH from 30.0 to 34.0 min, 10 mM KOH from 34.0 to 38.0 min, 100 mM KOH from 38.0 to 42.0 min, and 15 mM KOH from 42.0 to 45.0 min. The IC flow rate was 0.38 mL/min, supplemented postcolumn with 0.15 mL/min makeup flow of 0.01% FA in MeOH. The temperatures of the autosampler, column oven and ion suppressor were set at 10, 30 and 20 °C, respectively. The injector needle was automatically washed with water and 5 μL of each sample were loaded onto the column. The following ESI source settings were used: curtain gas, 20 arbitrary units; temperature, 400°C; ion source gas I, 60 arbitrary units; ion source gas II, 40 arbitrary units; collision gas, medium; ion spray voltage, -4500 V; declustering potential, -150 V; entrance potential, -10 V; and exit potential, -10 V. For scheduled selected reaction monitoring (SRM), Q1 and Q3 were set to unit resolution. The collision energy was optimized for each GroPInsP by direct infusion of the corresponding deacylated standard. The scheduled SRM detection window was set to 3 min, and the cycle time was set to 1.5 s. Data were acquired with Analyst version 1.6.2 (AB Sciex). Skyline (64-bit, 3.5.0.9319) was used to visualize results, integrate signals over the time, and quantify all lipids that were detected by MS.29

Preparation of human platelets. Blood from four individual healthy volunteers was collected to obtain four individual samples in ACD-buffer (70 mM citric acid, 116 mM sodium citrate, 111mM glucose, pH 4.6) and centrifuged at 200 g for 20 min. The obtained platelet-rich plasma was added to modified Tyrode-HEPES (N-2-hydroxyethyl-piperazone-N´2-ethanesulfonic acid) buffer (137 mM NaCl, 2 mM KCl, 12 mM NaHCO3, 5 mM glucose, 0.3 mM Na2HPO4, 10 mM HEPES, pH 6.5). After centrifugation at 900 g for 10 min and removal of the supernatant, the resulting platelet pellet was resuspended in Tyrode-HEPES buffer (pH 7.4, supplemented with 1 mM CaCl2). Platelet stimulation experiment. Freshly isolated and resuspended human platelets in 100 µL at a concentration of 1x106 platelets/µL were stimulated with either 0.01 U/mL thrombin or 1 μg/mL CRP for 5 min. After centrifugation for 5 min at 640 g at 25°C, the pellets were shock frozen in liquid nitrogen and stored at -80°C. Cell culture. Mesenchymal stem cells (OP9) were grown following a previously published protocol.27 Briefly, cells were grown in MEM with L-glutamine, 20% FBS, and 100 U/mL penicillin/streptomycin. Cultures were maintained at 37°C in humidified atmosphere with 5% CO2, and the medium was renewed every 4 days. After reaching 80% confluence, the cells were trypsinized, washed with PBS and collected from culture dish. The cells were aliquoted to 1x107 cells per sample, centrifuged at 400 g for 5 min, the supernatant was removed, and the cell pellet were snap frozen in liquid nitrogen. Membrane preparation from rat hippocampal brain tissue. Subcellular fractionation of rat hippocampus was performed as described earlier.28 3.5 g of rat hippocampal tissue was homogenized in 10 mL/g buffer A (0.32 M sucrose, 5 mM HEPES, pH 7.4) including protease inhibitor cocktail (PI) and phosphatase inhibitor (PhosSTOP) and centrifuged at 1 000 g for 10 min. The pellet was re-homogenized and centrifuged in buffer A. The resulting pellet 1 containing nuclei and cell debris was discarded and the supernatants were combined. The combined supernatants were centrifuged at 12 000 g for 20 min (Sorvall RC6, F13-14 x 50cy rotor). The pellet P2 was re-homogenized in buffer A and centrifuged as previously at 12 000 g for 20 min. The resulting pellet was collected as the hippocampus heavy membrane fraction.