Isotopic Labeling Procedure for Proteome Analysis

The isotopic labeling procedure was similar to that previously described by Zhang et al.^{35 (link)} The media utilized for isotopic labeling was Eagle’s Minimum Essential Medium (ATCC) supplemented with 15% dialyzed FBS (Thermo Scientific), 100 U/mL penicillin, and 100 U/mL streptomycin. Cells were gradually adapted to this media by replacing normal FBS with dialyzed FBS within five passages. Cells were then plated at a density of 1,000,000 cells per 10-cm plate.
One day after plating, the dividing cultures were switched to MEM labeling media for SILAC (Thermo Scientific) supplemented with l-arginine:HCl (¹³C6, 99%) and l-lysine:2HCl (¹³C6, 99%; Cambridge Isotope Laboratories) at concentrations of 0.1264 g/L and 0.087 g/L, 15% dialyzed FBS (Thermo Scientific), 100 U/mL penicillin, and 100 U/mL streptomycin. For whole proteome analysis, cells were collected after 0, 1, 2, 3, and 5 days of labeling and washed with PBS. For analysis of isolated PrP^Sc aggregates, cells were collected after 0, 1, 2, 3, and 4 days of labeling and washed with PBS. All cell pellets were frozen before further analysis.