Purified phosphopeptides and 300 μg of global peptides were reconstituted in 0.1% trifluoroacetic acid and fractionated on PierceTM High pH reversed-phase peptide fractionation spin columns using the vendor provided methodology (Thermo Fisher Cat No: A32993). Global TMT-peptides (flow through from TiO2 spin tips) were fractionated into 9 fractions, and TMT-labeled enriched phosphopeptides were fractionated into 6 fractions (including the 5% acetonitrile wash). Each fraction was dried by speed vacuum and reconstituted in 30 μL 0.1% formic acid (FA, CHEBI:30751).
