Fig. 1.

HR-SPROX as a tool for measuring protein stability. (A) Theoretical basis of HR-SPROX. Methionine residues (white squares) are oxidized by H₂O₂ to form methionine sulfoxides (red squares). Methionine oxidation rates differ between folded and unfolded states. Thus, protein denaturation can be quantified by measuring the extent of oxidation across different denaturant concentrations at a constant oxidation time (dashed line). The resulting denaturation curves can be used to measure four types of information (numbered 1–4): baseline oxidation, midpoint of denaturation ([denaturant]_1/2), m value, and free energy of folding (ΔG_folding or ΔG_f). For two-state proteins, the latter two parameters can be determined by fitting the denaturation curve to an equation derived from the linear extrapolation model (LEM) as described in the text. (B) HR-SPROX workflow. Cells are lysed under native conditions, and folded proteins (black ovals) are unfolded with increasing concentrations of GdmCl. Methionines are converted to methionine sulfoxides (red squares) by addition of H₂O₂. A control experiment lacking H₂O₂ is included and used as a normalization point. Extracts are digested into peptides, and each sample (corresponding to a different denaturant concentration) is labeled with a unique tandem mass tag (TMT) and subsequently combined and analyzed by LC-MS/MS. Reporter ion intensities at the MS2 level are internally normalized to create denaturation curves, monitoring either the increase in methionine sulfoxide-containing peptides or the decrease in unoxidized methionine-containing peptides.