Lipidomic Analysis of Extracellular Vesicles

Each of the TLC bands visualized from microvesicular or exosomal sample was scrapped from the silica plate, resuspended in 2:1(v/v) chloroform/methanol and analyzed using UltrafleXtreme MALDI-TOF/TOF mass spectrometer (Bruker Daltonik; Bremen, Germany) as previously described^{41 (link)}. Briefly, the TOF was calibrated using a peptide mixture (Bruker Daltonik) before acquisition of the sample spectra. Equal volume (1–2 µl) of lipid extract and MALDI matrix solution (0.5 M 2,5-dihydroxybenzoic acid in methanol containing 0.1% trifluoroacetic acid) was mixed in a 0.6-ml microcentrifuge tube. Then, 1 µl of the mixture was spotted onto a MALDI plate. The mass spectra were acquired over the m/z range of 0–2,000 in positive ion Reflectron TOF mode. The laser power was adjusted to a point just above the ionization threshold of the sample, and the laser rate was set at 10 Hz with 1,000 laser shots per acquisition. A total of 10 acquisitions were averaged for each sample.