10 plex isobaric tandem mass tags

The major fraction of lyophilized peptides (95%) were re-dissolved in 28.5% lactic acid (Sigma), 57% acetonitrile (LC-MS grade, Sigma), 0.28% TFA (Sigma), and applied to TiO₂ spin tips (88303, Thermo Pierce) for phosphopeptide enrichment according to manufacturer’s protocol. Eluted phosphopeptides were acidified with formic acid (pH 2-3), desalted using ZipTip C18 tips (100 μL, Millipore), and lyophilized. The two sets of peptides (phospho-enriched and unenriched) were separately labeled with 10-plex isobaric tandem mass tags (90406, Thermo Scientific) according to manufacturer’s protocol with slight modification. TMT reagents were reconstituted to 8 mg/mL in anhydrous acetonitrile (Sigma) and added to lyophilized peptides dissolved in 100 uL of 200 mM HEPES buffer, pH 8.0 (~8:1 reagent/peptide ratio). Labeling reaction was carried out in room temperature for 1 hr with gentle shaking, and quenched with 5 uL of 5% hydroxylamine (Thermo Scientific). Labeled peptides were combined into a single pool per experiment, acidified with formic acid (pH 2-3), desalted using ZipTip C18 tips (100 μL, Millipore), and lyophilized. The final processed peptides were dissolved in LC-MS/MS Buffer A (H₂O with 0.1% formic acid, LC-MS grade, Sigma Aldrich) for LC-MS/MS analysis.