USA, North America cat number B01020001) with 30 sec/30 sec on/off cycles for 15 minutes in a water bath at 4 °C. After subsequent centrifugation at 12,000 rpm for 20 min, protein concentrations were determined by Bradford protein assay (BioRad Cat No: 5000006). 400 μg equivalent of protein from each brain were then treated with 5 mM tris(2-carboxyethyl)phosphine hydrochloride (Sigma-Aldrich Cat No: C4706) to reduce disulfide bonds and the resulting free cysteine thiols were alkylated with 10 mM chloroacetamide (Sigma Aldrich Cat No: C0267). Samples were next diluted with 100 mM Tris.HCl (Sigma-Aldrich Cat No: 10812846001; to a final urea concentration of 2 M to carry out Trypsin/Lys-C based overnight protein digestion to derive peptides (1:100 protease:substrate ratio, Mass Spectrometry grade, Promega Corporation, Cat No: V5072.) (Levasseur et al. 2019; Li et al. 2020)
