Disulfide Mapping of a Recombinant Humanized IgG1 Antibody Containing Unpaired Cysteine

摘要

1. A Lys-C peptide mapping method was developed for monitoring the disulfide linkages in an IgG1 antibody containing an unusual number of Cys residues (34 Cys instead of 32). 2. NEM-alkylation prior to digestion was found necessary to prevent disulfide scrambling during sample preparation. 3. The optimized method requires 10-fold molar excess of NEM, 3-hr incubation in denaturing buffer at 37°C and pH 6.5, with NEM added prior to denaturation. 4. Eight expected disulfide-linked peptides were found by mass matching and further confirmed by reduction with DTT. 5. The presence of unpaired Cys at residue number 47 in the heavy chain was confirmed by MS and MS/MS analysis.