Ahmed, M.; Bellomio, P.; Manta, B.; Swovick, K.; Welle, K.; Hryhorenko, J.; Ghaemmaghami, S.

Oxidation of free methionine plays important roles in cellular redox homeostasis, yet its accurate quantification has been hindered by methodological challenges. Here, we introduce free Methionine Oxidation by Blocking (fMObB), a mass spectrometry-based method that enables accurate measurement of the fractional oxidation of free methionines. Applying fMObB to Escherichia coli, we quantify free methionine oxidation under basal and oxidative stress conditions, and in strains lacking methionine sulfoxide reductases. We find that during oxidative stress, free methionines exhibit higher oxidation levels than protein-bound methionines and that methionine sulfoxide reductases play a central role in maintaining reduced free methionine pools. Together, this work establishes fMObB as a generalizable strategy for probing free methionine redox states in cellular systems.