Peptides were desalted on Sep-Pak® Vac cartridges (WatersTM Cat No: WAT054955), dried down, and resuspended in 24 μL of 50 mM triethylammonium bicarbonate. Each sample was then labeled for two hours at room temperature, with 3 mg of Tandem Mass Tag Pro (TMTpro) reagent (16-plex kit, manufactures instructions Thermo Fisher Scientific,TMTpro™ Isobaric Label Reagent Set; Cat No: 44520, lot no. UL297970) (Li et al. 2020). Samples were randomized and labeled as indicated in the supplementary excel data files (https://github.com/dlhagger/AUD-Multi-Omics). Labelling reactions were quenched by adding 5% hydroxylamine to the reaction mixtures and holding at room temperature for 15 minutes. Labeled peptides were then mixed, desalted on 3 × 100 mg Sep-Pak® Vac (WatersTM Cat No: WAT036820) cartridges and dried by speed vacuum prior to phosphopeptide enrichment.
