The sulfur-containing amino acid methionine (Met) plays critical roles in protein synthesis, methylation, and sulfur metabolism. Both in its free form and in the form of an amino acid residue, it can be oxidized to the R and S diastereomers of methionine sulfoxide (MetO). Organisms evolved methionine sulfoxide reductases (MSRs) to reduce MetO to Met, with the MSRs type A (MSRA) and type B (MSRB) being specific for the S and R forms of MetO, respectively. In mammals, the selenoprotein MSRB1 plays an important protein repair function, and its expression is tightly regulated by dietary selenium. In this chapter, we describe a protocol for determining the concentration of protein-based Met-R-O and its analysis in HEK293 cells using a genetically encoded ratiometric fluorescent biosensor MetROx. We also describe the procedure for quantifying MSR activities in cell extracts using specific substrates and a reverse phase HPLC-based method.
Keywords: Fluorescent microscopy; Genetically encoded fluorescent sensors; HPLC; Methionine sulfoxide reductase; Oxidative stress; Protein oxidation.