Chromatography and mass spectrometry

The chromatographic separation was performed on an Acquity UPLC BEH300 C4 column (100 × 1.0 mm; 1.7 μm particle size; Waters Corporation) using a Waters Aquity UPLC system connected to an ABSCIEX 6500 QTRAP mass spectrometer for ion detection. All the samples were injected in duplicates and the flow rate was set to 100 μl/min. Solvent gradients were set, starting from 55% Solvent A (water + 0.1% formic acid)- 45% Solvent B (acetonitrile + 0.1% formic acid) from 0–5 min; then 45% B to 100% B from 5–10 min; 100% B was held from 10–15 min; between 15–16 min, 100% B was lowered to 45% B and held there till 20th min to re-equilibrate the column.

On the mass spectrometer, neutral loss scans were employed during pilot standardization experiments on biological samples to identify the parent ions that would lose neutral fragments corresponding to 490 a.m.u and 382 a.m.u—these were indicative of PIP₂ and PIP species respectively and likewise 155 a.m.u for PE species (Sharma et al, 2019). Thereafter, we quantified PIP, PIP₂, and PE species in biological samples using the selective MRM method in the positive ion mode. Area under the peaks was calculated via the Sciex MultiQuant software. For each run, area under the peak for each species of PIP₂, PIP, and PE was normalized to PIP₂, PIP and PE internal standard peak respectively. Thereafter, the sum of normalized areas for all the species of PIP₂ or PIP was taken and divided by the sum of normalized areas for all the species of PE in each of the biological samples to account for differences in total phospholipids extracted across samples. The MRM mass pairs used for PIP, PIP₂, and PE species identification and quantification are listed below in Table S4.

Table S4. List of multiple reaction monitoring values used for detection of the lipids.

The other mass spectrometer parameters were as follows:

ESI voltage: +5,100–5200 V; source temperature: 300°C, curtain gas (CUR): 35–37, ion source gas 1 (GS1):15–20, ion source gas 2 (GS2): 15–20.