| Autor: |
Michiels TJM; Division of BioTherapeutics, Leiden Academic Centre for Drug Research (LACDR), Leiden University, 2333 CC, Leiden, The Netherlands.; Intravacc, Institute for Translational Vaccinology, 3721 MA, Bilthoven, The Netherlands., Schöneich C; Department of Pharmaceutical Chemistry, The University of Kansas, Lawrence, Kansas 66047, United States., Hamzink MRJ; Intravacc, Institute for Translational Vaccinology, 3721 MA, Bilthoven, The Netherlands., Meiring HD; Intravacc, Institute for Translational Vaccinology, 3721 MA, Bilthoven, The Netherlands., Kersten GFA; Division of BioTherapeutics, Leiden Academic Centre for Drug Research (LACDR), Leiden University, 2333 CC, Leiden, The Netherlands.; Intravacc, Institute for Translational Vaccinology, 3721 MA, Bilthoven, The Netherlands., Jiskoot W; Division of BioTherapeutics, Leiden Academic Centre for Drug Research (LACDR), Leiden University, 2333 CC, Leiden, The Netherlands., Metz B; Intravacc, Institute for Translational Vaccinology, 3721 MA, Bilthoven, The Netherlands. |
| Abstrakt: |
Formaldehyde-inactivated toxoid vaccines have been in use for almost a century. Despite formaldehyde's deceptively simple structure, its reactions with proteins are complex. Treatment of immunogenic proteins with aqueous formaldehyde results in heterogenous mixtures due to a variety of adducts and cross-links. In this study, we aimed to further elucidate the reaction products of formaldehyde reaction with proteins and report unique modifications in formaldehyde-treated cytochrome c and corresponding synthetic peptides. Synthetic peptides (Ac-GDVEKGAK and Ac-GDVEKGKK) were treated with isotopically labeled formaldehyde ( 13 CH 2 O or CD 2 O) followed by purification of the two main reaction products. This allowed for their structural elucidation by (2D)-nuclear magnetic resonance and nanoscale liquid chromatography-coupled mass spectrometry analysis. We observed modifications resulting from ( i ) formaldehyde-induced deamination and formation of α,β-unsaturated aldehydes and methylation on two adjacent lysine residues and ( ii ) formaldehyde-induced methylation and formylation of two adjacent lysine residues. These products react further to form intramolecular cross-links between the two lysine residues. At higher peptide concentrations, these two main reaction products were also found to subsequently cross-link to lysine residues in other peptides, forming dimers and trimers. The accurate identification and quantification of formaldehyde-induced modifications improves our knowledge of formaldehyde-inactivated vaccine products, potentially aiding the development and registration of new vaccines. |
