Light-induced formation of singlet oxygen selectively oxidizes methionines in the weighty chain of IgG2 antibodies. that contains both Msr enzyme activities permitted the quantitative reduction of all Met(O) diastereomers. Using these Msr enzymes in combination with peptide mapping we were able to detect and differentiate diastereomers of methionine sulfoxide within the highly conserved heavy chain of an IgG2 that had been photo-oxidized as well as those in an IgG1 oxidized with peroxide. The quick recognition of the stereospecificity of methionine oxidation by Msr enzymes not only definitively differentiates Met(O) diastereomers which previously has been indistinguishable using traditional techniques but also provides an important tool that may contribute to understanding of the mechanisms of protein oxidation and development of fresh formulation strategies to stabilize protein therapeutics. ions (and ions efficiently localizes the +16 Da changes to the Met residue and discount rates changes at Trp or either of the His residues present in H35. Both oxidized peptides comprising M428 (Fig. 3C) showed related MS/MS fragmentation patterns indicating the presence of two stereomers of Met428. Differentiation of the stereomers by MS/MS was not possible because of the identical masses. Of the five Met residues with this IgG2 three were determined to be susceptible to photo-oxidation. These oxidation hotspots have been highlighted in B (M252) C (M428) and D (M397) of Number 3. The Dovitinib Dilactic acid ESR1 pub graphs in Number 5 show the level of oxidation for each Met residue like a function of exposure time to UV (A) or Xe (B) irradiation. Regardless of the light source Met 252 showed the highest level Dovitinib Dilactic acid of oxidation compared with that of Met397 and Met428. Relative oxidative susceptibility of the three Met residues was as follows: M252 > M428 > M397. For Met252 approximately 14% of the starting material at time zero was already oxidized to Met sulfoxide. This was consistently observed for different manufactured lots of this IgG2. Number 5 (A and B) Pub graphs comparing UV and Xe induced Met modifications in an IgG2. (C) Pub graph comparing the effect of adding free Met (10 mM) to UV and Xe (24 hour exposure) induced Met modifications in IgG2. A comparison of irradiation by UV to that by Xe indicated that UV irradiation resulted in a gradual increase in Met sulfoxide whereas Xe irradiation led to a rapid increase in oxidation in all three Met locations (Fig. 5A and B). The level of oxidation in the UV-exposed samples after 48 hours was related to that in the Xe-exposed samples after Dovitinib Dilactic acid 8 hours of exposure. All three Met residues were nearly (93% for M397) or fully oxidized in the 48 hour time point for Xe-exposed samples (Fig. 5B). Met252 was oxidized further to Met sulfone under these conditions. This might become attributed to the intensity of the Xe irradiation becoming greater than that of the UV thereby leading to more rapid oxidation in the Xe-exposed samples. Also as previously reported Met252 was the most labile of the three Met residues 14 which might facilitate oxidation of this residue from sulfoxide to sulfone. The relative area oxidized for samples formulated with 10 mM Met and samples formulated without Met after 24 hours irradiation by UV and Xe is usually shown in Physique 5C. For both light sources the presence of 10 mM Met was demonstrated to inhibit oxidation of the IgG2 Met residues by approximately half. This is consistent with previous reports that Met can be added to protein formulations as an anti-oxidant to limit protein oxidation. Reduction of met sulfoxide diastereomers at M428 in the IgG2. Since it was not possible to differentiate between the presumed methionine sulfoxide diastereomers by mass spectrometry Msr enzymes MsrA and MsrB were utilized for the identification of Met-S-(O) and Met-R-(O) respectively. The diastereomers of oxidized Dovitinib Dilactic acid Met made up of peptides M397(O) (Fig. 3D) and M428(O) (Fig. 3C) were separated whereas those from M252(O) were either not resolved or not present in the sample (Fig. 3B). The effect of MsrBA on M428(O) diastereomers is usually shown in Physique 6A. Both peaks were completely reduced after 8 hours incubation confirming the identification of an oxidized methionine residue in the peptide and excluding oxidation of other amino acids such as tryptophan or histidine. The second diastereomeric peak that eluted designated M428(O)2 was reduced successively with increasing incubation time with MsrA (Fig. 6B). In contrast.