Heterogeneity in Disulfide Bond Reduction in IgG1 Antibodies Is Governed by Solvent Accessibility of the Cysteines

• Published in: Antibodies (Basel) Vol. 12; no. 4; p. 83
• Main Authors: Natesan, Ramakrishnan, Dykstra, Andrew B, Banerjee, Akash, Agrawal, Neeraj J
• Format: Journal Article
• Language: English
• Published: Switzerland MDPI AG 01.12.2023
• Subjects: Accessibility; Acids; Alkylating agents; Alkylation; Analysis; Antibodies; Chemical bonds; Chromatography; Cysteine; differential alkylation; disulfide bond; Heterogeneity; Immunoglobulin G; Light chains; Liquid chromatography; Mass spectrometry; Mass spectroscopy; Methods; Molecular dynamics; Molecular weight; peptide mapping; Peptides; Physiological aspects; Reagents; Reduction; Residues; SASA; Scientific imaging; Solvents; United States; St Louis Missouri; United States > US; peptide mapping; disulfide bond; SASA; differential alkylation; molecular dynamics
• ISSN: 2073-4468; 2073-4468
• DOI: 10.3390/antib12040083

We studied unpaired cysteine levels and disulfide bond susceptibility in four different γ-immunoglobulin antibodies using liquid chromatography-mass spectrometry. Our choice of differential alkylating agents ensures that the differential peaks are non-overlapping, thus allowing us to accurately quantify free cysteine levels. For each cysteine residue, we observed no more than 5% to be unpaired, and the free cysteine levels across antibodies were slightly higher in those containing lambda light chains. Interchain and hinge residues were highly susceptible to reducing stresses and showed a 100-1000-fold higher rate of reduction compared to intrachain cysteines. Estimations of the solvent-accessible surface for individual cysteines in IgG1, using an implicit all-atom molecular dynamics simulation, show that interchain and hinge cysteines have >1000-fold higher solvent accessibility compared to intrachain cysteines. Further analyses show that solvent accessibility and the rate of reduction are linearly correlated. Our work clearly establishes the fact that a cysteine's accessibility to the surrounding solvent is one of the primary determinants of its disulfide bond stability.