Raman quantitative analysis of methionine oxidation and its promotion effect on dissolution of phase-separated liquid droplets of Pbp1

• Raman microscopy was used to quantitatively analyze Pbp1 LC droplets formed by LLPS. • Oxidation reduced the Pbp1 LC concentration in the droplet and caused its dissolution. • Methionine oxidation in Pbp1 LC was directly observed. • The link between the Pbp1 LC concentration and the oxidation in the droplet was shown. • The thresholds of the Pbp1 LC concentration and the oxidation for LLPS were evaluated. The Raman spectra of droplets of the low-complexity domain of an RNA-binding protein (Pbp1 LC) were measured to investigate the mechanism of the disappearance of Pbp1 LC droplets in oxidative environments. Raman quantification analysis indicated that the Pbp1 LC concentration in the droplets decreased with the addition of hydrogen peroxide. It was directly confirmed from the methionine band that the methionine residues were oxidized by hydrogen peroxide. We quantitatively evaluated the relationship between the degree of the methionine oxidation and the Pbp1 LC concentration in the droplets and showed that the droplet dissipation is induced by the methionine oxidation.