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. 2023 Jun 25;26(7):107205. doi: 10.1016/j.isci.2023.107205

Figure 1.

Figure 1

Identification of the YMD-oxidized derivatives using mass spectrometry and HPLC profile of its oxidation potential

(A) Schematic representation of the theoretical Met-oxidized forms of the YMD peptide. ∗ designates experimentally identified peptide variants in this work.

(B) LC-MS identification of the YMD peptide Met-oxidized derivatives from the standard peptide (upper panel) and from an acidic cell surface elution of A375 melanoma cells, stably expressing tyrosinase (A375+Tyr): extracted ion chromatograms for the native (N – m/z 516.21226), Met2 sulfoxide (M2SO – m/z 524.20973), Met6 sulfoxide (M6SO – m/z 524.20973), and the sulfoxide at Met2 and Met6 (M2SOM6SO – m/z 532.20717) of the YMD peptide. Retention time (min) and identity are assigned to each peak. The calculated mass of each peak corresponds to an increase of 16 and respective 32 Da compared to the native form.

(C) The standard peptide was subject to a series of oxidation reactions with several concentrations of hydrogen peroxide as described under STAR methods. An aliquot of 1 μg of peptide was analyzed using reversed phase high-performance liquid chromatography (RP-HPLC) for the separation of the oxidized products (left panel). Peak integration was performed for the annotated peaks N, M2SO, M6SO, and M2SOM6SO at each peroxide concentration. At 120 mM, ∼15% of the peptide is still detected as native, while at concentrations of 600 mM and higher, the peptide is almost completely oxidized (right panel). Points are mean of triplicate replicates and error bars are SEM.

(D) Similar to C, but the oxidation reaction was monitored in time (left panel) to derive the kinetics of the reaction (right panel). It can be observed that after 60 min of incubation with hydrogen peroxide, less than 10% native form was detected. Inset: a linear correlation (R2: 0.9766) was found between the log of the native peptide remained fraction and the reaction time suggesting a pseudo-first-order reaction ([N]0 and [N]t are the concentrations of the reduced Met at the initial t0 time and time t).