Identification of Methionine Sulfoxide Diastereomers in Immunoglobulin Gamma Antibodies Using Methionine Sulfoxide Reductase enzymes
During prolonged periods of storage methionines in antibodies and other proteins are known to become oxidized to form methionine sulfoxides and sulfones. While these post-translational modifications are commonly identified by peptide mapping, it is currently problematic to identify the relative abundances of the S- and R-diastereomers of methionine sulfoxide (Met(O)) due to their identical polarities and masses. Accordingly, we have developed a separation methodology for the rapid and quantitative determination of the relative abundances of Met(O) diastereomers. Identification of these diastereomers takes advantage of the complementary stereospecificities of methionine sulfoxide reductase (Msr) enzymes MsrA and MsrB, which respectively promote the selective reduction of S- and R-diastereomers of Met(O). In addition, an MsrBA fusion protein that contained both Msr enzyme activities permitted the quantitative reduction of all Met(O). Using these Msr enzymes in combination with peptide mapping we are able to detect and differentiate Met-diastereomers in a monoclonal IgG2 and IgG1 antibody. We also monitored the formation of sulfones and studied the rate of oxidation in the different Met residues in our IgG2 antibody. The reported ability to separate and identify diastereomers of Met(O) permits a more complete characterization of Met oxidation products. All the affected Met residues (M251, M427, M396) in this study are conserved in human IgG sequences and therefore offer predictive potential in characterizing oxidative modification.
- Research Organization:
- Pacific Northwest National Lab. (PNNL), Richland, WA (United States)
- Sponsoring Organization:
- USDOE
- DOE Contract Number:
- AC05-76RL01830
- OSTI ID:
- 991988
- Report Number(s):
- PNNL-SA-70209; TRN: US201021%%445
- Journal Information:
- mAbs, 2(3):299-308, Vol. 2, Issue 3; ISSN 1942-0862
- Country of Publication:
- United States
- Language:
- English
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Related Subjects
60 APPLIED LIFE SCIENCES
ANTIBODIES
ENZYMES
IMMUNOGLOBULINS
METHIONINE
MODIFICATIONS
OXIDATION
OXIDOREDUCTASES
PEPTIDES
PROTEINS
RESIDUES
STORAGE
SULFONES
SULFOXIDES
immunoglobulin gamma antibody
methionine sulfoxide
oxidation
photo-oxidation
methionine sulfoxide reductase