Peptide Purification and Tandem Mass Tagging

We performed peptide purification and labeling according to previously published methods (Grecco et al., 2021a (link); Grecco et al., 2022a (link)). To summarize our previous methods, digestion was halted by addition of 0.3% final v/v trifluoroacetic acid (TFA), and peptides were desalted on Waters Sep-Pak^® Vac cartridges (Waters™ Cat No: WAT054955) with a wash of 1 mL 0.1% TFA followed by elution in 0.6 mL of 70% acetonitrile 0.1% formic acid (FA). Peptides were dried by speed vacuum and resuspended 50 mM triethylammonium bicarbonate. Peptide concentrations were checked by Pierce Quantitative colorimetric assay (Cat No: 23275). The same amount of peptide from each sample was then labeled for 2 h at room temperature, with 0.5 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. VI310352) (Li et al., 2020 (link)). Labeling reactions were quenched with 0.3% hydroxylamine (final v/v) at room temperature for 15 min. Labeled peptides were then mixed and dried by speed vacuum. The TMT-labeled peptide mix was desalted to remove excess label using a 100 mg Waters SepPak cartridge, eluted in 70% acetonitrile, 0.1% formic acid and lyophilized to dryness.