Fig. 2. Development of a chemoproteomic assay for direct and isoform-specific evaluation of native ERK activity. (A) Biochemical assays for measuring recombinant ERK1 and ERK2 activity. Substrate assays can measure individual ERK isoforms but are limited to assaying of recombinant proteins. (B) Western blots can measure native ERK1/2 activity but readouts are indirect and often cannot discern isoform specificity of inhibitors. Phosphorylation of ERK1/2 and downstream substrates are biomarkers used to evaluate cellular activity of compounds. (C) Schematic of a chemoproteomic assay to measure native ERK activity in an isoform-specific fashion. Measurement of ERK1 and ERK2 in complex proteomes enables parallel evaluation of potency and selectivity. Conserved lysines in the active-sites of kinases react with acyl phosphate groups of ATP activity-based probes to covalently modify active kinases with desthiobiotin tags for quantitative LC-MS/MS analysis. (D) MS1 extracted ion chromatogram (EIC) of a peptide m/z from chemoproteomic analysis of mouse brain proteomes resulted in ambiguous identification of 2 LC-resolved peptides that matched ERK1/2 (labeled RT1 and RT2). (E, F) MS2 spectra resulting from fragmentation of the same peptide m/z for each LC peak (RT1 and RT2). The spectra showed an identical fragmentation pattern from each chromatographically separated peak. Red amino acid indicates probe-modified lysine residue. Green X indicates an ambiguous isoleucine/leucine isomer in ERK active-site peptides that cannot be distinguished from MS2 spectra.