K8 lysine

RPE-1 WT (control) cells were grown in heavy (K8) SILAC DMEM (DMEM with high glucose minus glutamine, lysine, and arginine) supplemented with 10% dialyzed FBS (Gibco), 50 µg/mL K8 lysine (Cambridge Isotope Labs), 85 µg/mL arginine (Sigma-Aldrich), and 2 mM glutamine/1 mM sodium pyruvate (Thermo Fisher). RPE-1 cells trisomic for chromosome 12 or 21 were grown in light (K0) SILAC DMEM (same formulation as above, with 50 µg/mL K0 lysine; Sigma-Aldrich). Cells were cultured for eight generations prior to testing K8 labeling efficiency by MS and then expanded for harvest at passage 10. Prior to harvest, cells were treated with 25 µM chloroquine for 16 h followed by addition of 1 µM MG-132 (Calbiochem) for 6 h to induce protein aggregation. Cells were harvested by trypsinization, neutralized in SILAC DMEM, and washed with PBS; an equal number of heavy and light cells were mixed (WT + Ts12; WT + Ts21), treated with sodium azide, and snap-frozen for aggregate purification.