PhosB was solubilized in equilibration buffer (10 mM potassium phosphate, pH 7.0) and diluted 1:1 (vol/vol) by the addition of ice-cold quench buffer (500 mM glycine-HCl, pH 2.35). Measurements were performed in three subsequent runs with a standard wash runs, i.e. pepsin wash (1.6 M guanidinium-HCl, 4% acetonitrile, 0.8% formic acid), in between. Then, a blank run (1:1 mix of equilibration and quench buffer) was performed and carry-over was evaluated based on peptide intensities. Detailed LC settings are provided in Supporting Information (Table and).