Sample Preparation for Mass Spectrometry

Sample preparation for MS was performed according to our laboratory protocols modified from previous publications (30 , 31 (link), 32 (link)). To prepare enriched samples for MS analysis, EV fractions and cells were lysed in 8 M urea containing protease and phosphatase inhibitors. Hundred micrograms of protein from the cell lysates and the entire volume of the EV lysates were then reduced with 1 mM DTT at RT for 30 min and alkylated with 5 mM iodoacetamide in the dark for 30 min with rotation. Proteins were digested overnight with 2 μg of lysyl (Lys-C) endopeptidase (Wako, 127-06621) at RT on shaker. Samples were then diluted (7-fold) with 50 mM ammonium bicarbonate to bring the urea concentration to 1 M. Samples were then digested overnight with 2 μg of trypsin (Thermo Fisher Scientific, 90058) at RT on shaker. The resulting peptide solutions were acidified to a final concentration of 1% formic acid (FA) and 0.1% TFA, desalted with a HLB columns (Waters, 186003908), and dried down in a vacuum centrifuge (SpeedVac Vacuum Concentrator).