Measurement of site-specific H2O2-sensitivity and identification of sulfenic

In vitro H2O2-sensitivity of CLOCK cysteines was measured with a quantitative chemoproteomic approach termed as QTRP (Quantitative Thiol Reactivity Profiling) as previously reported. In brief, 5 μg of recombinant CLOCK proteins were reduced with 1 mM DTT for 30 min at room temperature. After reaction, excess DTT was removed by Zeba™ Spin Desalting Column (Thermo Scientific) pre-equilibrated with PBS, pH 7.4. Reduced CLOCK proteins were then reacted with or without H2O2 (150 μM) for 10 min at room temperature. The reaction was quenched with catalase. Proteins were labeled with 100 μM thiol-reactive IPM probe for 30 min at room temperature. The reversibly oxidized cysteines were reduced with 1 mM DTT at 75°C for 15 min and alkylated with 5 mM IAM for 30 min in the dark at the room temperature.