PhosB was solubilized in equilibration buffer (10 mM potassium phosphate, pH 7.0) and diluted 1:1 (vol/vol) with the quench buffer (100 mM potassium phosphate, pH 2.3). The HDX manager of the nanoACQUITY system was equipped with a Vanguard column (BEH C18, 130 Å, 1.7 m, 2.1 mm x 5 mm; Waters) and an Acquity UPLC column (BEH C18, 130 Å, 1.7 m, 1.0 mm x 100 mm; Waters) for peptide trapping and separation, respectively. Protein digestion was performed online with the UPLC chromatographic system using an in-house packed protease column (immobilized pepsin agarose resin) at 15 C. The generated peptides were trapped and washed with solvent A at 200 L/min for 3 minutes. Subsequently, peptides were separated by applying a 7.5-minute linear gradient from 8 to 35% solvent B at 40 L/min. Peptides were measured in positive ion mode between 50 and 2,000 m/z on the Xevo G2-XS mass spectrometer. Experiments were performed in triplicates on the standard two-valve and extended three-valve configuration applying standard bottom-up HDX-MS workflow. Detailed LC settings are provided in Supporting Information (Table and).