Pa 17 0 17 0

Saturated and unsaturated FFAs were obtained from Sigma-Aldrich. Phospholipid standards (PC 17:0/17:0, PE 17:0/17:0, PS 17:0/17:0, PA 17:0/17:0, PG 17:0/17:0, PC 12:0/13:0, PE 17:0/14:1, PS 17:0/14:1, PA 12:0/13:0 and PG 12:0/13:0) were purchased from Avanti Polar Lipids. 1,1-carbonydiimidiazole, 1,4 dioxane, triethylamine, iodomethane, iodomethane-d₃, iodoethane, formic acid, citric acid, disodium hydrogen phosphate ammonium formate, acetonitrile, 1-butanol, methanol and chloroform were purchased from Sigma Aldrich. All reagents were analytical grade or equivalent. All lipid extractions were performed in 2 mL polypropylene LoBind safe-lock tubes (Eppendorf).

Lipids were extracted from wandering 3^rd instar larval fat body as previously described [60 (link)]. The lipidomic analyses were carried out on an analytical system comprising an Agilent HPLC 1260 coupled with a SCIEX 5500 QTRAP. Separation of individual classes of polar lipids by normal phase HPLC was carried out using a Phenomenex Luna 3u silica column (i.d. 150x2.0 mm). Multiple reaction monitoring (MRM) transitions were set up for quantitative analysis of various polar lipids. Individual lipid species were quantified by referencing to spiked internal standards. PC-14:0/14:0, LPC-C20, PE-14:0/14:0, PS-14:0/14:0, PA-17:0/17:0, PG-14:0/14:0 were obtained from Avanti Polar Lipids and dioctanoyl phosphatidylinositol (PI, 16:0-PI) was obtained from Echelon Biosciences, Inc. Separation of glycerol lipids (DAG and TAG) by reverse phase HPLC/ESI/MS/MS was carried out on a Phenomenex Kinetex 2.6μ-C18 column (i.d. 4.6x100mm). Using neutral loss-based MS/MS techniques, the levels of TAG were calculated as relative contents to the spiked d5-TAG 48:0 internal standard (CDN Isotopes), while DAG species were quantified using 4ME 16:0 Diether DG as an internal standard (Avanti Polar Lipids). Free cholesterols and ergosterols were analyzed using HPLC/APCI/MS/MS with the corresponding d6-Cho (CDN Isotopes) as the internal standard.

Polar lipids were analyzed using an Agilent 1260 HPLC system coupled with a triple quadrupole/ion trap mass spectrometer (5500Qtrap; SCIEX) as described previously39 (link). Separation of individual lipid classes of polar lipids by normal phase (NP)-HPLC was carried out using a Phenomenex Luna 3μ-silica column (internal diameter 150 × 2.0 mm) with the following conditions: mobile phase A (chloroform: methanol: ammonium hydroxide, 89.5:10:0.5) and mobile phase B (chloroform: methanol: ammonium hydroxide: water, 55: 39: 0.5: 5.5). MRM transitions were set up for comparative analysis of various polar lipids. Individual lipid species were quantified by referencing to spiked internal standards. PC-14:0/14:0, PE-14:0/14:0, PS-14:0/14:0, PA-17:0/17:0, PG-14:0/14:0, LPA-17:0 and LPC-17:0 were obtained from Avanti Polar Lipids (Alabaster, AL) and LIPID MAPS.