Proteomic Profiling of Human Brain Regions

Gray matter tissue (~20 mg) from DLPFC and NAc were collected from fresh-frozen coronal tissue blocks via cryostat to minimize contamination from white matter and other subregions [29 (link), 30 (link)]. Homogenate and synaptosome preparations were obtained using a variation of our enrichment protocol for postmortem human brain tissues [24 (link), 31 (link), 32 ] with SynPER reagent (ThermoFisher). From each sample, 10 µg total protein (as measured by Micro BCA) was reduced, alkylated, and trypsin digested on S-Trap™ micro spin columns (ProtiFi). Subject pairs were randomly assigned to TMT blocks and labeled with TMTPro channels 1–10, with brain regions and preparations assigned to separate blocks [33 ]. Additional aliquots from each sample were used for a pooled control, digested separately with S-Trap Midi™ columns, divided then labeled with TMTPro channels 1 and 12. TMT labeled preparations from the same block were pooled with 10 µg of the labeled pooled controls. The TMT labeled peptide pools were separated into eight fractions with the Pierce™ High pH Reversed-Phase Peptide Fractionation Kit (ThermoFisher Scientific), evaporated, and reconstituted in 20 µl 97% H₂O, 3% ACN, 0.1% formic acid.