Figure 2.

Identification of YMD oxidative modifications from the HPLC fractions and their purity estimation

(A) Theoretical b and y ions detectable following CID fragmentation of the std. YMD peptide. The diagnostic ions, flanking the two Met residues, used in our analysis are labeled in green.

(B) Schematic reaction of the Met sulfoxide side-chain neutral loss during CID fragmentation. Sulfoxide peptides can lose methanesulfenic acid following CID fragmentation rendering −64 Da neutral loss species.

(C) MS/MS fragmentation pattern obtained by CID fragmentation of the mass corresponding to the Met6 sulfoxide version (M6_SO – upper panel) and the native peptide (N – lower panel). Pink lines correspond to the theoretical b and y ions obtained by in silico fragmentation of M6_SO, respective native peptide. Black lines denote the m/z of the ions detected following fragmentation and blue and red peaks map the b and y ions. Green peaks denote the ions flanking the two Met residues (Met2 and Met6). The green arrows above the MS/MS spectra also indicate these ions.

(D) Extracted ion chromatograms (EIC) corresponding to the precursor ions (left panel) and product ions flanking the two Met residues for the M6_SO version. For both precursor and product ions, the mass accuracy is indicated and the corresponding retention time.

(E) Similar as in C, but for the Met2 sulfoxide version (M2_SO).

(F) Similar as in D, but for the M2_SO.

(G) Similar as in C, but for sulfoxide version of both Met residues (M2_SOM6_SO).

(H) Similar as in D but for M2_SOM6_SO.

(I) UV-VIS HPLC trace at 275 nm assesed on a Hypersil Gold preparative column cat no 25005-109070, ThermoFisher Scientifc of the fractions corresponding to the native (N-first panel), Met2 sulfoxide (M2_SO - second panel), Met6 sulfoxide (M6_SO – third panel), Met2 and Met6 sulfoxide versions (M2_SOM6_SO – fourth panel).