Cell Type-Specific Proteomics from Brain Tissue

To perform cell type-specific proteomics and phosphoproteomics, astrocytes and neurons were separated from cortical single-cell suspensions as previously described^{62 (link)} with slight modifications. Single-cell suspensions were incubated with FcR Blocking Reagent (Miltenyi Biotec, 130-092-575) and Anti-ACSA-2 MicroBeads (Miltenyi Biotec, 130-097-678) for 10–15 min at 2–8 °C. Cells were spun down for 10 min at 300 × g at room temperature and resuspended in a minimal volume of PBS with 0.5% of BSA before passing through a LS Column. Flow-through was collected for further neuron separation, the LS Column that retained astrocytes was removed from the magnetic field and the ACSA-2 labeled astrocytes were eluted. Cells were pelleted from flow-through at 300 × g, 8 min, the supernatant carefully removed, and resuspended in a minimal volume of PBS with 0.5% of BSA. Suspensions were incubated with Non-Neuronal Cell Biotin-Ab cocktail (Miltenyi Biotec, 130-115-389) for 5 min and Anti-Biotin MicroBeads (Miltenyi Biotec, 130-090-485) for 10 min at 2–8 °C, respectively. Following incubation, suspensions were passed through a LS Column placed in a magnetic field and non-labeled neuronal cells were collected in flow-through.