Characterizing Thermal Transitions of IgG with Mass Spectrometry.

ABSTRACT: Variable temperature electrospray ionization (ESI) is coupled with mass spectrometry techniques in order to investigate structural transitions of monoclonal antibody immunoglobulin G (IgG) in a 100-mM ammonium acetate (pH 7.0) solution from 26 to 70 °C. At 26 °C, the mass spectrum for intact IgG shows six charge states +?22 to +?26. Upon increasing the solution temperature, the fraction of low-charge states decreases and new, higher-charge state ions are observed. Upon analysis, it appears that heating the solution aids in desolvation of the intact IgG precursor. Above ~?50 °C, a cleavage event between the light and heavy chains is observed. An analysis of the kinetics for these processes at different temperatures yields transition state thermochemistry of ?H‡?=?95?±?10 kJ mol-1, ?S‡?=?8?±?1 J mol-1 K-1, and ?G‡?=?92?±?11 kJ mol-1. The mechanism for light chain dissociation appears to involve disulfide bond scrambling that ultimately results in a non-native Cys199-Cys217 disulfide bond in the light chain product. Above ~?70 °C, we are unable to produce a stable ESI signal. The loss of signal is ascribed to aggregation that is primarily associated with the remaining portion of the antibody after having lost the light chain. Graphical Abstract.

SUBMITTER: Brown CJ 

PROVIDER: S-EPMC6866664 | biostudies-literature | 2019 Nov

REPOSITORIES: biostudies-literature