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.我们培养了分别转染野生型（WT）或FH型超氧化物歧化酶（SOD1）的NSC-34细胞。随后采用含蛋白酶抑制剂的RIPA缓冲液裂解细胞（设置三个复孔），将裂解液样品分别与靶向VAPB、VCP或Stathmin-2的抗体进行孵育。于4℃条件下孵育过夜后，加入Protein G磁珠，将混合体系置于室温孵育2小时。之后借助磁力架将磁珠与上清液分离，并用质谱级1×PBS洗涤磁珠三次。最后移除磁珠上残留的全部液体，将磁珠置于-80℃冷冻保存，随后寄送至密歇根大学开展磁珠上胰蛋白酶消化、搭载电子转移解离（ETD）与场不对称离子迁移谱（FAIMS）的OrbiTrap Ascend Tribrid质谱仪的90分钟鸟枪法质谱蛋白质组学分析，以及使用Proteome Discoverer v3.0软件进行数据分析。同时设置对照样品组，包含仅细胞裂解液与未结合任何抗体的磁珠，以排除磁珠介导的非特异性结合干扰。质谱分析完成后，对对照组、Stathmin-2、VAPB及VCP样品的数据集进行空值清除处理。随后采用t检验对突变型样品与对照组开展统计学比较，为每个蛋白质生成对应的p值。