Middle-Down Fragmentation for the Identification and Quantitation of Site-Specific Methionine Oxidation in an IgG1 Molecule
In: Journal of pharmaceutical sciences, Jg. 99 (2010), Heft 11, S. 4469-4476
academicJournal
- print, 18 ref
Zugriff:
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. Middle-down 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.
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Middle-Down Fragmentation for the Identification and Quantitation of Site-Specific Methionine Oxidation in an IgG1 Molecule
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Autor/in / Beteiligte Person: | PIPES, Gary D ; CAMPBELL, Phillip ; BONDARENKO, Pavel V ; KERWIN, Bruce A ; TREUHEIT, Michael J ; GADGIL, Himanshu S |
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Zeitschrift: | Journal of pharmaceutical sciences, Jg. 99 (2010), Heft 11, S. 4469-4476 |
Veröffentlichung: | Washington, DC; Hoboken, NJ: American Pharmaceutical Association, Wiley, 2010 |
Medientyp: | academicJournal |
Umfang: | print, 18 ref |
ISSN: | 0022-3549 (print) |
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