Brain Proteome Extraction and TMT Labeling

Mice were euthanized and their brains rapidly dissected from the skull. Brains or cells were homogenized in SDS-containing Lysis Buffer (2% SDS, 62.5 mM HEPES/NaOH, pH 8.0; preheated to 90°C), with the aid of 1.0 mm zirconia beads and a Mini-BeadBeater-8 (Biospec Products Inc., Oklahoma, USA). Following three cycles of 1 minute bead beading, the lysates were further incubated at 90°C to deactivate residual enzymatic activities in the extracts. Protein levels were adjusted by BCA colorimetric assay (Thermo Scientific, Nepean, Ontario, Canada) before sample preparation for global proteome analyses. Protein precipitation, denaturation, reduction, alkylation and digestion were performed as previously described [7 (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.