Kras is a molecular switch controlling the activity of the MAPK pathway. As such, it determines ERK activation state. In this study we identified many novel p-sites that were regulated upon KRAS inhibition. Many of these regulated p-sites were located within a SP/TP motif, the recognition motif of proline directed Kinases such as ERK. In this study we used parallel reaction monitoring (PRM) to determine the ability of ERK to phosphorylate synthetic peptides carrying a subset of these p-sites in a time-resolved manner. We identified that ERK was able to phosphorylate 10 of the tested sequences of which only one was phosphorylated on a mutated control peptide.

Synthetic peptides were ordered from JPT (15-mers or 16-mers). Peptide design was based on acquired DDA phosphoproteomic data that showed regulation of the specific sites after KRAS inhibition. In total 19 p-sites were synthesized as spike tides and controls included for those that carried an additional STY within the synthesized sequence (with Alanine in the central position). Reconstituted phospho-peptides were pooled, and a subtraction of the pool was removed (TP 0/ - ERK). Recombinant ERK2 (Reaction biology) was added to the peptide pool, and subfractions were removed and quenched after several incubation time points (0 min, 5 min, 15 min, 30 min, 60 min, 90 min, 120 min, 180 min). The sample was desalted and measured via PRM on a Thermo Fusion Lumos utilizing the provided transition list.

Synthesized phospho-peptides incubated with recombinant ERK2 for 0, 5, 15, 30, 60, 90, 120, and 180 minutes.