Finally, we compared the “NCys-ome” between cells under normoxia and hypoxia. As expected, we again saw RGS4/5, but now also >10 other NCys that were seemingly regulated in their abundance or reactivity under hypoxia; only two were from known NCys-containing proteins!

We then validated the probes by knocking out the NCys oxidase ADO, which should stabilise NCys-bearing proteins such as RGS4 and RGS5. Using an live-cell ABPP approach, we showed that the oxSTEF probes could indeed fish these two out!

First, we used an “unbiased” amino acid profiling workflow in FragPipe inspired by @stephanhacker2.bsky.social. We indeed found the expected NCys conjugate by open searches, and localized it to NCys using offset searches. Using oxSTEF6 we identified >800 NCys in lysates from cancer cells!

We then realized that no one had tried to use a chemical probe to fish for these – and so the idea of using the oxSTEF-probes as NCys ABPP-probes was born💡

At a group meeting I then asked: “Does NCys exist in live cells?” No one knew, so I went back to the office to research 📖 I found literature describing how NCys are (seemingly) uncommon but dictates protein stability (via N-degron pathways) depending on the oxidation state: Oxidation = degradation!

Background: We previously developed the oxSTEF probes for disulphide rebridging in bioconjugation contexts. The probes feature two sulfur-based leaving groups which can exchange for other nucleophiles. Interestingly, with calcitonin we observed quite good NCys reactivity and selectivity.

In my first #bsky post, I am very happy and proud to share the new(ish) preprint of my post doc project on N-terminal cysteine (NCys) chemoprotomics using the oxSTEF probes!

Preprint at 👉 www.doi.org/10.26434/chemrxiv-2024-4w549

Skeeturial (??) below ⬇️ #ChemSky #ChemBio