Fig. 5 RP-isoDTB workflow for quantitative MS1 and MS2 (PRM) analysis. (a) Intact cells of P. aeruginosa were treated with DMSO or 10 µM dynorphin A (Dyn), lysed in a buffer containing the nucleophilic HA-yne probe (125 mM) at pH 4 and clicked to the isoDTB tags. Modified proteins were combined, tryptically digested, enriched on streptavidin beads, eluted from the beads and subjected to LC-MS/MS analysis. (b) Waterfall plot representing the ratio between dynorphin A (light) and  DMSO (heavy) treated HA-yne modified Asp and Glu residues. Red dots indicate sites, that also have UniProt annotated pAsp sites. (c) PRM transitions (Dyn/light vs. DMSO/heavy) of pAsp annotated and HA-yne modified peptides of response regulators CprR and ParR. Data was analyzed using the Skyline software. MS2 ratios of 20.8 and 2.0 were obtained for CprR and ParR, respectively, unraveling CprR as the only protein with highly enhanced pAsp modification.