Fig. 3.

Quantitative oxidation of methionine-containing peptides. A, peptides containing Met residues can be observed by LC-MS in the unoxidized, Met sulfoxide, or Met sulfone forms when modified by zero, one, or two oxygens, respectively. B, controlled oxidation of the N-terminal MQIF peptide from Ub. Quantitative conversion of unoxidized Met to Met sulfoxide is observed in 0.01% H₂O₂, 5% FA after 30 min and remains stable out to 21.5 h. The maximal signals for unoxidized and Met sulfone-containing peptides were normalized based upon Met sulfoxide signal (0.01% H₂O₂; 30 min) based on the observation that Met sulfoxide conversion was essentially complete under these conditions. Further oxidation to Met sulfone is observed beginning at concentrations of ∼1% H₂O₂, 5% FA for 30 min, whereas complete conversion was only seen at higher concentrations and with longer incubation times. C and D, controlled oxidation of the GGMQ linear polyUb signature peptide and K6 isopeptide-linked -GG signature peptide reveals similar kinetics to MQIF with Met sulfoxide observed with 0.01% H₂O₂, 5% FA after 30 min and increasing amounts of Met sulfone observed at higher concentrations and longer times. E, signal intensities for a selection of Ub peptides (unbranched and -GG signature) show consistent signal intensity across the range of oxidation conditions used. Values represent the mean area under the curve for n = 3 at each data point.