The desalted and pTyr peptide–depleted flow-through was fractionated by high-pH reversed-phase (RP) LC. Protein digest (4 mg) was loaded onto an LC system consisting of an Agilent 1200 high-performance LC with mobile phases of 5 mM NH4HCO3 (pH 10) (A) and 5 mM NH4HCO3 in 90% MeCN (pH 10) (B). The peptides were separated by a 4.6 mm × 250 mm Zorbax Extend-C18, 3.5 μm, column (Agilent) over 96 min at a flow rate of 1.0 ml/min by the following timetable: hold 0% B for 9 min, gradient from 0 to 10% B for 4 min, 10 to 28.5% B for 50 min, 28.5 to 34% B for 5.5 min, 34 to 60% B for 13 min, hold at 60% B for 8.5 min, 60 to 0% B for 1 min, reequilibrate at 0% B for 5 min. Fractions were collected at 1-min intervals from 0 to 96 min by the shortest path by row in a 1-ml deep well plate (Thermo Fisher Scientific). The high-pH RP fractions were concatenated into 24 samples by every other plate column starting at 15 min (for example, sample 1 contained fractions from wells B10, D10, F10, etc.). The remaining fractions were combined such that fractions from 12 to 14 min were added to sample 1, all fractions after 86 min were added to sample 24, and all fractions from 0 to 11 min were combined into sample “A.” Ninety-five percent of every 12th fraction of the 24 samples was combined (1,13; 2,14;…) to generate 12 more samples, which were dried down and stored at −80°C before phosphopeptide enrichment by IMAC.

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