Middle-down fragmentation for the identification and quantitation of site-specific methionine oxidation in an IgG1 molecule
✍ Scribed by Gary D. Pipes; Phillip Campbell; Pavel V. Bondarenko; Bruce A. Kerwin; Michael J. Treuheit; Himanshu S. Gadgil
- Publisher
- John Wiley and Sons
- Year
- 2010
- Tongue
- English
- Weight
- 391 KB
- Volume
- 99
- Category
- Article
- ISSN
- 0022-3549
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✦ Synopsis
A middle-down LC/MS approach, for the rapid quantitation and characterization of site-specific methionine oxidation in a recombinant monoclonal IgG1 molecule, is described. An IgG1 antibody was digested with endoprotease LysC under limited proteolytic conditions to produce two major components; an antigen binding fragment (Fab) and a crystallizable fraction (Fc). These fractions were then reduced to produce three major species; light chain (LC), Fc/2 which is the C terminal region of the heavy chain (HC) and the N-terminal heavy chain region (Fd). These three fragments were separated by reversed-phase HPLC using a diphenyl column. The diphenyl column resolved site-specific methionine oxidation in all three subunits. Middledown N-terminal sequencing with a LCT premier mass spectrometer was used to identify the sites of oxidation in the LC. Sites of oxidation in the Fc/2 were identified using middle-down collision-induced dissociation (CID) on a Qtof premier. This method allowed for the rapid quantitation and identification of oxidation on each methionine residue in an IgG1 molecule.