DOI: 10.6084/m9.figshare.27650253
title: Untargeted metabolomics Raw data of EVs from LLC-Pri cells and LLC-LM cells

description: <b>Sample Collection:</b> Serum-free medium was collected from the cultures of cells after 24 h incubation. Extracellular vesicles (EVs) were isolated using a sequential centrifugation. Briefly, cells, dead cells and cell debris were first removed through centrifugation at 500 x g for 10 min, 3000 × g for 10 min and 10,000 × g for 30 min at 4 °C. The supernatant was then collected and underwent ultracentrifugation at 100,000 × g for 70 min at 4 °C. After removing the supernatant, crude extract of the EVs in precipitation were washed with PBS at 100,000 × g for another 70 min. The final pellet was resuspended in PBS for further experiments. <b>Extraction:</b> Take EV samples stored at -20 °C (including sample preparation QC), and place them in a 4 °C refrigerator to thaw them until no ice cubes are visible in the samples. Add 100µL of each sample (including QC) to the EP tube, then freeze the remaining samples. Add 700µL of extractant containing internal standard 1 (methanol: acetonitrile: water, 4:2:1, V/V/V), shake for 1min, and place in -20°C refrigerator for 2h. Centrifuge 25000 x g, 4 °C for 15 min. Move samples out of centrifuge and transfer 600 µL of the supernatant to a split new EP tube. Use drying machine to dry. Add 180µL methanol: pure water (1:1 v/v), vortex for 10min, until all dissolved in the reconstituted solution. Centrifuge 25000 x g, 4 °C for 15 min. Take the supernatant into a new EP tube. Take 20 μL of each sample and mix into QC samples. Pass prepared supernatant to LC-MS/MS steps.<b>Chromatography:</b> Chromatographic separation was performed on a Waters ACQUITY UPLC BEH C18 column (1.7 μm, 2.1 mm × 100 mm, Waters, USA), and the column temperature was maintained at 45 °C. The mobile phase consisted of 0.1% formic acid (A) and acetonitrile (B) in the positive mode, and in the negative mode, the mobile phase consisted of 10 mM ammonium formate (A) and acetonitrile (B). The gradient conditions were as follows: 0-1 min, 2% B; 1-9 min, 2%-98% B; 9-12 min, 98% B; 12-12.1 min, 98% B to 2% B; and 12.1-15min, 2% B. The ﬂow rate was 0.35 mL/min and the injection volume was 5 μL.<b>Mass spectrometry:</b> Using Q Exactive (Thermo Fisher Scientific, USA) perform primary and secondary mass spectrometry data acquisition. The full scan range was 70‒1050 m/z with a resolution of 120,000 and the automatic gain control (AGC) target for MS acquisitions was set to 3e6 with a maximum ion injection time of 100 ms. Top 3 precursors were selected for subsequent MSMS fragmentation with a maximum ion injection time of 50 ms and resolution of 30,000, the AGC was 1e5. The stepped normalized collision energy was set to 20, 40 and 60 eV. ESI parameters were setting as : Sheath gas flow rate was 40, Aux gas flow rate was 10, positive-ion mode Spray voltage (KV) was 3.80, negative-ion mode Spray voltage (KV) was 3.20, Capillary temperature was 320°C, Aux gas heater temperature was 350°C.<b>Data transformation and metabolite identification:</b> After importing the off-line data of mass spectrometry into compound discoverer 3.3 (Thermo Fisher Scientific, USA) software and analyzing the mass spectrometry data in combination with bmdb (BGI metabolome database), mzcloud database and chemspider online database, a data matrix containing information such as metabolite peak area and identification results will be obtained. Software information: Compound Discoverer Version v.3.3. Parameter: Parent ion mass deviation: &lt; 5ppm Mass deviation of fragment ions: &lt; 10ppm Retention time deviation: &lt; 0.2min Offical Website: https://mycompounddiscoverer.com/