Conformational studies of a monoclonal antibody, IgG1, by chemical oxidation: structural analysis by ultrahigh-pressure LC-electrospray ionization time-of-flight MS and multivariate data analysis.
We describe the development of a method in which protein oxidation by H2O2 followed by ultrahigh-pressure liquid chromatography (UHPLC) coupled with electrospray ionization time-of-flight mass spectrometry (ESI-ToFMS) and multivariate analysis are used to detect alterations in conformational states of proteins. In the study reported here, an IgG1 monoclonal antibody in native and denatured conformational states was oxidized by treatment with hydrogen peroxide. Peptide fragments generated by tryptic digestion were then analyzed by UHPLC-ESI-ToFMS. After reducing noise and extracting peaks from the LC-MS data using MzExplorer, software developed in-house and based on Matlab, we were able to distinguish peptides arising from the native and denatured states of the oxidized protein by principal component analysis. Peptides containing residues, which are inclined to undergo oxidation, such as methionine, are founded to be particularly important in this approach. We believe that the methodology could facilitate attempts to characterize the conformational states of recombinant monoclonal antibodies and other proteins.
Zamani
Leila
L
Department of Analytical Chemistry, Stockholm University, SE-10691 Stockholm, Sweden.
Andersson
Fredrik O
FO
Edebrink
Per
P
Yang
Yang
Y
Jacobsson
Sven P
SP
D000911:Antibodies, Monoclonal / Q000737:chemistry*; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D006861:Hydrogen Peroxide / Q000737:chemistry; D007074:Immunoglobulin G / Q000737:chemistry*; D008715:Methionine / Q000737:chemistry; D015999:Multivariate Analysis; D010084:Oxidation-Reduction; D011487:Protein Conformation; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*
D000911:Antibodies, Monoclonal; D007074:Immunoglobulin G; D008715:Methionine; D006861:Hydrogen Peroxide