Speedvac vacuum concentrator

Sample preparation for MS was performed according to our laboratory protocols modified from previous publications^{50 (link)–52 (link)}. To prepare enriched samples for mass spectrometry analysis, EV fractions and cells were lysed in 8M urea containing protease and phosphatase inhibitors. 100 μg of protein from the cell lysates and the entire volume of the EV lysates were then reduced with 1 mM dithiothreitol (DTT) at room temperature for 30 min and alkylated with 5 mM iodoacetamide (IAA) 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 (ABC) to bring the urea concentration to 1 M. Samples were then digested overnight with 2 μg of trypsin (Thermo, 90058) at RT on shaker. The resulting peptide solutions were acidified to a final concentration of 1% formic acid (FA) and 0.1% triflouroacetic acid (TFA), desalted with a HLB columns (Waters, 186003908), and dried down in a vacuum centrifuge (SpeedVac Vacuum Concentrator).

Briefly, samples (wet weight: ∼50 mg) were homogenized, followed by tissue protein extraction, reduction, alkylation, and trypsin. Tissue samples were thawed on ice and weighed before lysis. Tissue samples were broken down and homogenized in 8 M urea (dissolved in PBS (PBS, Wisent, Nanjing, China)) supplemented with protease inhibitor (Biotools, Shanghai, China) by using magnetic lysis beads (Full Moon BioSystems, CA, USA). The supernatant was collected after centrifugation with 12, 000 g at 4 °C. Protein concentrations were determined by using the Bicinchoninic Acid (BCA) method. A pooled sample with equal aliquots taken from control and disease samples was prepared to serve as the internal control. 50 μg of each sample was used for in-solution digestion. Proteins were reduced with 5 mM dithioreitol (DTT, INALCO SPA MILANO, Italy) and alkylated with 12.5 mM iodoacetamide (IAM, Sigma-Aldrich Inc., MO, USA) in the dark at room temperature. The protein solution was diluted to 1.5 M urea by using PBS and digested by using trypsin protease (Promega, Madison, WI, USA) of a 100:1 protein to protease ratio for 16 h at 37 °C. The tryptic peptides were desalted by using Sep-Pak columns (Waters, MA, USA) and dried by using a SpeedVac Vacuum concentrator.