Estimation of the deamidation rates of major deamidation sites in a Fab fragment of mouse IgG1-? by capillary isoelectric focusing of mutated Fab fragments.
The deamidation of asparagine (Asn or N) residues in proteins is a common post-translational chemical modification. The identification of deamidation sites and determination of the degree of deamidation have been carried out by the combination of peptide mapping and mass spectrometry. However, when a peptide fragment contains multiple amides, such analysis becomes difficult and sometimes impossible. In this report, a quantitative method for estimating the deamidation rate of a specific amide in a protein is presented without using peptide mapping. Five Asn residues of a recombinant fragment antigen binding (rFab) (mouse IgG1, ?) were mutated to a serine (Ser) residue, one by one, through site-directed mutagenesis, and the single-residue deamidation rates of the original rFab and the mutants were determined using capillary isoelectric focusing. The difference of the rate between the original rFab and the mutant was assumed to be equal to the deamidation rate of the specific Asn residue, which had been mutated. Among five mutants established, three major deamidation sites-H chain Asn135, L chain Asn157, and L chain Asn161, using the Kabat numbering system-were identified, accounting for 66%, 29%, and 7% of the single-residue deamidation of the original rFab, respectively. Although the former two have been known by peptide mapping, the last one, which resides on the same tryptic peptide that carries one of the former two, previously has not been identified. For the first time, the deamidation rate constants of the three sites were estimated to be 10.5 × 10(-3) h(-1), 4.6 × 10(-3) h(-1), and 1.1 × 10(-3) h(-1) in 0.1 M phosphate buffer, pH 7.5 at 37 °C, respectively, with corresponding half-life of 2.8 days, 6.3 days, and 27 days. The method should be applicable to any recombinant proteins.