Global Proteome Analysis of 1C11 Cells

1C11 and PrP^null-1C11 cells were homogenized in SDS-containing lysis buffer (2% SDS, 62.5 mM HEPES/NaOH, pH 8.0; preheated to 90°C), with the use of 1.0 mm zirconia beads and a Mini-BeadBeater-8 (Biospec Products Inc., USA). Following three cycles of 1 min bead beading, the lysates were further incubated at 90°C to deactivate residual enzymatic activities in the extracts. Protein levels were adjusted by the bicinchoninic acid method (BCA) colorimetric assay (Thermo Scientific, Canada) before sample preparation for global proteome analyses. Protein precipitation, denaturation, reduction, alkylation, and digestion were performed as in [79 (link)]. MS grade trypsin was from Thermo Scientific. Tryptic peptides were covalently modified with the TMTsixplex isobaric label reagent set (Thermo Scientific) according to the protocol supplied by the manufacturer. The quantitative mass spectrometry was performed as in [48 (link)].