Mass spectrometry analysis of in vitro nitration of a recombinant human IgG1 monoclonal antibody.
Nitration of a recombinant human monoclonal antibody was carried out in vitro by incubating the antibody with the nitrating reagent tetranitromethane (TNM). The susceptible sites of nitration were identified using high-performance liquid chromatography/mass spectrometry (HPLC/MS). In general, tyrosine residues in the variable domains of the antibody are more susceptible to nitration, while tyrosine residues in the constant domains are relatively resistant to nitration. However, one tyrosine residue in the CH1 domain and one tyrosine residue in the CH2 domain are highly susceptible to nitration. Interestingly, the susceptible tyrosine residue in the CH2 domain is followed by the conserved asparagine residue that is glycosylated.
Process Sciences Department, Abbott Bioresearch Center, 100 Research Drive, Worcester, MA 01605, USA. hongcheng.liu@abbott.com
Hongcheng
H
Liu
Georgeen
G
Gaza-Bulseco
Chris
C
Chumsae
Czeslaw H
CH
Radziejewski
D000911:Antibodies, Monoclonal / Q000032:analysis*; D002853:Chromatography, Liquid; D006020:Glycopeptides / Q000032:analysis; D006031:Glycosylation; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000032:analysis; D007141:Immunoglobulin Fc Fragments / Q000032:analysis; D007074:Immunoglobulin G / Q000032:analysis*; D007143:Immunoglobulin Heavy Chains / Q000032:analysis; D007147:Immunoglobulin Light Chains / Q000032:analysis; D007202:Indicators and Reagents; D013058:Mass Spectrometry; D008970:Molecular Weight; D009566:Nitrates / Q000737:chemistry*; D010446:Peptide Fragments / Q000737:chemistry; D010449:Peptide Mapping; D011994:Recombinant Proteins / Q000032:analysis; D013774:Tetranitromethane / Q000737:chemistry; D014357:Trypsin / Q000737:chemistry; D014443:Tyrosine / Q000032:analysis
D000911:Antibodies, Monoclonal; D006020:Glycopeptides; D007140:Immunoglobulin Fab Fragments; D007141:Immunoglobulin Fc Fragments; D007074:Immunoglobulin G; D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains; D007202:Indicators and Reagents; D009566:Nitrates; D010446:Peptide Fragments; D011994:Recombinant Proteins; D014443:Tyrosine; D014357:Trypsin; D013774:Tetranitromethane