Middle-down fragmentation for the identification and quantitation of site-specific methionine oxidation in an IgG1 molecule.
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.
Pipes
Gary D
GD
Department of Process and Product Development, Amgen, Inc., USA.
Campbell
Phillip
P
Bondarenko
Pavel V
PV
Kerwin
Bruce A
BA
Treuheit
Michael J
MJ
Gadgil
Himanshu S
HS
D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid / Q000191:economics / Q000379:methods; D004355:Drug Stability; D007074:Immunoglobulin G / Q000737:chemistry*; D013058:Mass Spectrometry / Q000191:economics / Q000379:methods*; D008715:Methionine / Q000737:chemistry*; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D011994:Recombinant Proteins / Q000737:chemistry
D007074:Immunoglobulin G; D011994:Recombinant Proteins; D008715:Methionine