HK2 lipidomics INFg
Description
HK2 cells were transfected with siRNA targeting ACSL5 and scrambled, for 24 h than incubated for additionnal 48 hours.
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For Lipidomics analysis, 100,000 HK-2 cells were spiked with 4.56 μL of internal standard lipid mixture containing 500 pmol of Chol-d6, 100 pmol of Chol-16:0-d7, 100 pmol of DAG 17:0-17:0, 50 pmol of TAG 17:0-17:0-17:0, 100 pmol of SM 18:1;2-12:0, 30 pmol of Cer 18:1;2-12:0, 30 pmol of GalCer 18:1;2-12:0, 50 pmol of LacCer 18:1;2-12:0, 300 pmol of PC 17:0-17:0, 50 pmol of PE 17:0-17:0, 50 pmol of PI 16:0-16:0, 50 pmol of PS 17:0-17:0, 30 pmol of PG 17:0-17:0, 30 pmol of PA 17:0-17:0, 40 pmol of Gb3 18:1;2-17:0, 25 pmol of GM3 18:1;2-18:0-d5, 25 pmol of GM2 18:1;2-18:0-d9, 25 pmol of GM1 18:1;2-18:0-d5, 30 pmol of LPA 17:0, 30 pmol of LPC 12:0, 30 pmol of LPE 17:1 and 30 pmol of LPS 17:1 and subjected to lipid extraction at 4 °C, as described elsewhere 35 . Briefly, the sample was dissolved in 200 μL of 155 mM ammonium bicarbonate and then extracted with 1 mL of chloroform-methanol (10:1) for 2 hours. The lower organic phase was collected, and the aqueous phase was re-extracted with 1 mL of chloroform-methanol (2:1) for 1 hour. The lower organic phase was collected and evaporated in a SpeedVac vacuum concentrator. Lipid extracts were dissolved in 100 μL of infusion mixture consisting of 7.5 mM ammonium acetate dissolved in propanol:chloroform:methanol [4:1:2 (vol/vol)]. Samples were analyzed by direct infusion in a QExactive mass spectrometer (Thermo Fisher Scientific) equipped witha TriVersa NanoMate ion source (Advion Biosciences). 5 µL of sample were infused with gas pressure and voltage set to 1.25 psi and 0.95 kV, respectively. DAG, TAG and CE species were detected in the 10:1 extract, by positive ion mode FTMS as ammonium aducts by scanning m/z= 580–1000 Da, at Rm/z=200=280 000 with lock mass activated at a common background (m/z=680.48022) for 30 seconds. Every scan is the average of 2 micro-scans, automatic gain control (AGC) was set to 1E6 and maximum ion injection time (IT) was set to 50ms. For FA profiling of DAG and TAG, a parallel reaction monitoring was performed with an inclusion list of m/z=580-1000 Da, at NCE of 20 and Rm/z=200=17500 for 90 seconds. Every scan is the average of 2 micro-scans, automatic gain control (AGC) was set to 1E5 and maximum ion injection time (IT) was set to 64 ms. PC, PCO, Cer, GlcCer, LPC and LPCO were detected as acetate adducts while PG, PE, PEO, LPE and LPEO were detected as deprotonated adducts in the 10:1 extract, by negative ion mode FTMS, after polarity switch by scanning m/z= 420–1050 Da, at Rm/z=200=280 000 with lock mass activated at a common background (m/z=529.46262) for 30 seconds. E All data was acquired in centroid mode. All lipidomics data were analyzed with the lipid identification software, LipidXplorer 36. Tolerance for MS and identification was set to 2 ppm.