Novel extracellular vesicle release pathway facilitated by toxic superoxide dismutase 1 oligomers

We grew NSC-34 cells transfected with either WT or FH SOD1. We lysed the cells (RIPA buffer with protease inhibitors, triplicate wells) and combined samples of the lysate with antibodies for either VAPB, VCP, or Stathmin-2. After incubating at 4°C overnight we added Protein G Magnetic Beads and allowed the mixture to incubate at room temperature for two hours. We then separated the beads from the supernatant using a magnet, and washed the beads three times with mass spectrometry grade 1X PBS. Finally, all liquid was removed from the beads and they were frozen at -80 °C before shipping them to the University of Michigan for on-bead trypsin digestion, 90-minute shotgun Mass Spectrometry proteomics using an OrbiTrap Ascend Tribrid with ETD and FAIMS, and analysis using Proteome Discoverer v3.0. Control samples including cell lysate and beads without any antibody are also analyzed to eliminate any non-specific binding to the magnetic beads. Following mass spectrometry analysis, the datasets for control, Stathmin-2, VAPB, and VCP samples were cleaned by removing empty values. Subsequently, a statistical t-test was employed to compare mutant samples against the control, generating p-values for each protein.