Nature research paper: Insulin-regulated serine and lipid metabolism drive peripheral neuropathy
Quantification of sphingolipid metabolites was determined using triple quadrupole liquid chromatography–mass spectrometry platform . Sphingolipid species were separated on a C8 column as previously described. Mobile phase A was composed of 100% HPLC-grade water containing 2 mM ammonium formate and 0.2% formic acid and mobile phase B consisted of 100% methanol containing 0.2% formic acid and 1 mM ammonium formate. The flow rate was 0.5 ml min.
. After extraction, a 50-µl aliquot was taken for protein quantification using BCA protein assay , and the remaining extract was spun for 10 min at 21,000at 4 °C. The supernatant was then transferred into a glass vial, and chromatographic separation and compound identification performed using Q Exactive Orbitrap MS with a Vanquish Flex Binary UHPLC system on an iHILIC- Classic, 150 mm by 2.1 mm, 5-mm particle, 200-Å column at 45 °C.
Chromatographic separation and lipid species identification was performed using Q Exactive orbitrap mass spectrometer with a Vanquish Flex Binary UHPLC system equipped with an Accucore C30, 150 × 2.1 mm, 2.6 µm particle column at 40 °C. Five microlitres of sample was injected. Chromatography was performed using a gradient of 40:60 v/v water: acetonitrile with 10 mM ammonium formate and 0.1% formic acid and 10:90 v/v acetonitrile: propan-2-ol with 10 mM ammonium formate and 0.
Lipids were analysed in positive mode using spray voltage 3.2 kV. Sweep gas flow was 1 arbitrary units, auxiliary gas flow 2 arbitrary units and sheath gas flow 40 arbitrary units, with a capillary temperature of 325 °C. Full mass spectrometry (scan range 200–2,000
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