High pressure liquid chromatography system

Pooled samples were fractionated on 1,290 infinity High Pressure Liquid Chromatography system (Agilent, Santa Clara, CA, USA) by injecting 900 μl of sample reconstituted in solvent A (10 mM TEABC in water; pH 8.5) on Xbridge (4.6 × 250 mm, 5 μm; Waters, Milford, MA, USA) column. The peptides were separated by using a gradient of 2% solvent A (10 mM TEABC in water; pH 8.5) to 40% solvent B (10 mM TEABC in 90% acetonitrile, pH 8.5) over 40 min and further taken to 100% solvent B where it was held for 5 min before coming back to 2% solvent A for re-equilibration (27 (link)). A total of 96 fractions were collected and concatenated to 10 fractions for total proteomic and 13 fractions for phosphoproteomic analysis. All the fractions were dried under vacuum, desalted using C18 StageTips and stored at −20°C till further analysis.

SEC analysis was performed according to a previously developed method (Turner et al., 2018 (link)). Briefly, all samples were analyzed on an Agilent high pressure liquid chromatography system using isocratic elution (100 mmol/L sodium phosphate supplemented with 250 mmol/L sodium chloride, pH 6.8) at 0.30 ml/min and monitored at 280 nm. 60 µg of antibody sample was injected onto a Waters Acquity UPLC Protein BEH SEC column (1.7 μm particle size, 200 Å pore size, 4.6 × 150 mm length).