Novel Formaldehyde-Induced Modifications of Lysine Residue Pairs in Peptides and Proteins: Identification and Relevance to Vaccine Development.

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 (13CH2O or CD2O) 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.