biostudies-literature00470Brown CJNational Institutes of HealthNIH HHSNIGMS NIH HHS2438-2445https://www.ebi.ac.uk/biostudies/studies/S-EPMC6866664biostudies-literatureUnknown30(11)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 ΔH^‡ = 95 ± 10 kJ mol^-1, ΔS^‡ = 8 ± 1 J mol^-1 K^-1, and ΔG^‡ = 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.Journal of the American Society for Mass SpectrometryCharacterizing Thermal Transitions of IgG with Mass Spectrometry.PMC68666645R01GM117207-045R01GM121751-02R01 GM117207R01 GM121751El-Baba TJClemmer DEWoodall DWBrown CJ47falseCharacterizing Thermal Transitions of IgG with Mass Spectrometry.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 ΔH^‡ = 95 ± 10 kJ mol^-1, ΔS^‡ = 8 ± 1 J mol^-1 K^-1, and ΔG^‡ = 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.2019-01-01T00:00:00Z2019 Nov2024-11-07T07:56:21.549Z2020-11-03T08:04:26ZS-EPMC68666643136398910.1007/s13361-019-02292-6