Identification of methionine sulfoxide diastereomers in immunoglobulin gamma antibodies using methionine sulfoxide reductase enzymes.
Light-induced formation of singlet oxygen selectively oxidizes methionines in the heavy chain of IgG2 antibodies. Peptide mapping has indicated the following sensitivities to oxidation: M252 > M428 > M397. Irrespective of the light source, formulating proteins with the free amino acid methionine limits oxidative damage. Conventional peptide mapping cannot distinguish between the S- and R-diastereomers of methionine sulfoxide (Met[O]) formed in the photo-oxidized protein because of their identical polarities and masses. We have developed a method for identification and quantification of these diastereomers by taking advantage of the complementary stereospecificities of the methionine sulfoxide reductase (Msr) enzymes MsrA and MsrB, which promote the selective reduction of S- and R-diastereomers of Met(O), respectively. In addition, an MsrBA fusion protein 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 rapid identification 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 new formulation strategies to stabilize protein therapeutics.
Khor
Hui K
HK
Department of Pharmaceutics, Amgen, Inc., Thousand Oaks, CA, USA.
Jacoby
Michael E
ME
Squier
Thomas C
TC
Chu
Grace C
GC
Chelius
Dirk
D
D000595:Amino Acid Sequence; D006801:Humans; D007141:Immunoglobulin Fc Fragments / Q000032:analysis / Q000378:metabolism; D007074:Immunoglobulin G / Q000737:chemistry* / Q000378:metabolism; D008715:Methionine / Q000031:analogs & derivatives* / Q000032:analysis / Q000378:metabolism; D051150:Methionine Sulfoxide Reductases / Q000737:chemistry; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D010449:Peptide Mapping / Q000379:methods*; D011994:Recombinant Proteins / Q000032:analysis; D016415:Sequence Alignment; D013237:Stereoisomerism
D007141:Immunoglobulin Fc Fragments; D007074:Immunoglobulin G; D011994:Recombinant Proteins; D008715:Methionine; D051150:Methionine Sulfoxide Reductases; C027219:methionine sulfoxide reductase; C013111:methionine sulfoxide
J Pharm Sci. 1997 Nov;86(11):1250-5
9383735
DNA. 1981;1(1):11-8
6299662
J Chromatogr B Analyt Technol Biomed Life Sci. 2007 May 1;850(1-2):285-94
17182291
Free Radic Biol Med. 2000 Nov 15;29(10):986-94
11084287
Annu Rev Immunol. 2000;18:739-66
10837074
J Biol Chem. 2002 Apr 5;277(14):12016-22
11812798
J Invest Dermatol. 2006 May;126(5):1128-34
16514415
Biochim Biophys Acta. 2005 Jan 17;1703(2):239-47
15680232
Biochemistry. 2007 Dec 11;46(49):14153-61
17997579
Biochemistry. 2002 May 21;41(20):6245-52
12009885
J Biol Chem. 2001 Dec 28;276(52):48915-20
11677230
Protein Sci. 2009 Feb;18(2):424-33
19165723
Monogr Allergy. 1986;19:40-51
2429173
J Am Soc Mass Spectrom. 2009 Mar;20(3):525-8
19103498
J Immunol. 1980 Sep;125(3):1048-54
6774012
J Biol Chem. 1947 Aug;169(3):477-91
20259080
J Immunol. 1989 Sep 1;143(5):1656-62
2527269
Proc Natl Acad Sci U S A. 2001 Nov 6;98(23):12920-5
11606777
Biochim Biophys Acta. 2005 Jan 17;1703(2):111-9
15680219
Biopolymers. 2000;55(4):288-96
11169920
J Am Chem Soc. 2004 Dec 22;126(50):16601-7
15600366
Nat Biotechnol. 2002 Nov;20(11):1118-23
12368813
Arch Biochem Biophys. 1999 Mar 1;363(1):43-54
10079116
Anal Biochem. 1996 Apr 5;236(1):56-62
8619496
FEBS Lett. 1999 Jul 23;455(3):247-50
10437782
Anal Chem. 2004 Nov 1;76(21):6374-83
15516130
J Pharm Sci. 2001 Mar;90(3):253-69
11170019
J Exp Med. 1990 Apr 1;171(4):1333-45
2139103
Arch Biochem Biophys. 1983 May;223(1):271-81
6859861
Anal Chem. 2004 Jul 15;76(14):3908-22
15253624
Proc Natl Acad Sci U S A. 1968 Dec;61(4):1414-21
5249817
J Pharm Sci. 2004 Dec;93(12):3096-102
15514984
Mol Immunol. 2009 May;46(8-9):1878-82
19269032
J Pharm Biomed Anal. 2000 Jan;21(6):1099-128
10708395
Mol Immunol. 2008 Feb;45(3):701-8
17719636
AAPS J. 2009 Sep;11(3):432-4
19499344
Nucleic Acids Res. 1986 Feb 25;14(4):1779-89
3081877
Proc Natl Acad Sci U S A. 1969 May;63(1):78-85
5257969
J Invest Dermatol. 2006 May;126(5):947-9
16619011
Biochim Biophys Acta. 2005 Jan 17;1703(2):135-40
15680221
Biochemistry. 2008 May 6;47(18):5088-100
18407665