PhosB was digested on the in-house packed protease column at a flow rate of 200 L/min. The generated peptides were collected for 1 minute and subsequently freeze dried for five hours. Peptides were resuspended in deuterated labeling buffer (10 mM potassium phosphate, pH read 6.6; 100% final D 2 O content) for four hours. The reaction was quenched by adding 1:1 (vol/vol) ice-cold quench buffer (500 mM glycine-HCl, pH 2.35). Measurements were performed in triplicates on both standard two-valve and extended three-valve configuration. Peptide trapping and separation was performed on standard C18 trap and analytical columns at 0 C. The protease column was replaced by a union and the compartment was kept at 15 C. The phospholipid trap column was kept on ice. Detailed LC settings are provided in Supporting Information (Table and).