Fig. 3. Discovery and identification of isomeric active-site peptides of mammalian ERKs. (A) Comparison of mouse and bird (Taeniopygia guttata) brain proteomes by chemoproteomic and western blot analyses. Birds lack the ERK1 gene, which enables distinction between the identities of ERK peptides detected from the mouse brain proteome. The elution time for the ERK2 peptide identified from bird brain matched the late-eluted peptide in mouse brain, which supports RT1 and RT2 as ERK1 and ERK2, respectively. MS2 fragmentation spectra of the bird brain ERK2 peptide was identical to that of the mouse brain ERK1 and ERK2 counterparts (see Fig. S2†). The presence of a single ERK in bird brain proteomes was confirmed by western blot analysis. (B) Synthetic peptides corresponding to non-probe modified ERK1 and ERK2 active site-sequences, differing by isoleucine (I) versus leucine (L), were analyzed to confirm elution profiles. The identical MS2 fragmentation spectra of the synthetic ERK1 (-LLI-, top) and ERK2 (-LLL-, bottom) peptides and similar LC elution profile are consistent with the results obtained for endogenous mouse brain ERK1 and ERK2 probe-modified peptides (see Fig. 2). Green amino acids highlight isoleucine/leucine isomers in ERK active site-sequences.