Domain-level stability of an antibody monitored by reduction, differential alkylation, and mass spectrometry analysis.
Human immunoglobulin G1 (IgG1) contains 12 domains, and each has an intrachain disulfide bond that connects the two layers of antiparallel beta-sheets. These intrachain disulfide bonds are shielded from solvents under native conditions. Therefore, accessibility of the disulfide bonds to reduction under conditions that unfold antibody has the potential to be a good indicator of the thermodynamic stability of each domain. The stability of a recombinant monoclonal antibody at the domain level was investigated using a novel method involving reduction of the disulfide bonds in the presence of increasing amounts of guanidine hydrochloride and alkylation with [(12)C]iodoacetic acid, which was followed by reduction of the remaining disulfide bonds and alkylation with [(13)C]iodoacetic acid. The percentage of modification by [(12)C]iodoacetic acid of each cysteine residue was calculated using mass spectra of the cysteine-containing tryptic peptides and used to follow the unfolding of each domain. It demonstrated that the CH2 domain was the least stable domain of the antibody, whereas the CH3 domain was the most stable domain of the antibody. Other domains showed intermediate resistance to the denaturant concentration, similar to the overall unfolding transition monitored by the intrinsic tryptophan fluorescence wavelength shift.
Process Sciences Department, Abbott Bioresearch Center, Worcester, MA 01605, USA. hongcheng.liu@abbott.com
Hongcheng
H
Liu
Chris
C
Chumsae
Georgeen
G
Gaza-Bulseco
Eric R
ER
Goedken
D000478:Alkylation; D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000235:genetics; D002851:Chromatography, High Pressure Liquid; D004220:Disulfides / Q000737:chemistry; D019791:Guanidine / Q000737:chemistry; D006801:Humans; D007074:Immunoglobulin G / Q000737:chemistry; D019807:Iodoacetic Acid / Q000737:chemistry; D013058:Mass Spectrometry / Q000379:methods*; D010084:Oxidation-Reduction; D011489:Protein Denaturation; D055550:Protein Stability; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics
D000911:Antibodies, Monoclonal; D004220:Disulfides; D007074:Immunoglobulin G; D011994:Recombinant Proteins; D019791:Guanidine; D019807:Iodoacetic Acid