Figure 5.

Inhibitor reactivity and redox state of KRAS^G12Cin vitro and in NIH 3T3 cells.A, as shown by mass spectrometry, prereduced KRAS^CCLW (as used in Fig. 3) reacts rapidly with ARS-853 (second panel, incubated 5 min with a 10-fold excess of the inhibitor). Oxidation of KRAS^CCLW by reaction with H₂O₂ (50-fold excess of H₂O₂ added for 10 min) produced a mixture of sulfenic acid (-SOH; confirmed by reaction with dimedone) and sulfinic acid (SO₂H) forms, which did not react with ARS-853. B, experimental workflow for H₂O₂ treatment and digestion of NIH 3T3 cells expressing HA-tagged KRAS^G12C (mass spectrometry analysis method). Created with Biorender.com. C, quantification of KRAS^G12C redox state in lysates from NIH 3T3 cells treated with control vehicle (PBS) or H₂O₂ (100 and 1000 μM). Representative extracted ion chromatograms of the KRAS C12-containing tryptic peptide labeled with NEM (representing reduced protein), d5-NEM (representing reversibly oxidized protein), or oxidized irreversibly to sulfinic acid (-SO₂H). Peak area quantification based on mass spectrometry PRM analysis shows the contribution of dominant fragment ions. NEM: N-Ethylmaleimide, PRM: Parallel Reaction Monitoring; d₅-NEM, N-ethyl-d₅-maleimide.