Maleimide-thiol chemistry is widely used in the synthesis of protein–protein conjugate vaccines. In this reaction, thiol and maleimide groups, either in the carrier protein or in the antigen molecules, are linked via a thiosuccinimide linker. The assignment of the conjugation sites is key and is usually done by LC-MS/MS analysis of the conjugate proteolytic digestion, resulting in a very complex mixture of linear peptides and cross-linked peptide pairs. During proteolysis, the thiosuccinimide linker is converted to its hydrolyzed and transcyclized forms. The set of MS/MS spectra assigned to cross-linked peptide pairs contains valuable information about the conjugation sites that must be validated using objective criteria for more reliable assignment. The extracted ion chromatograms (XICs) have not previously been considered for these validation purposes. In the LC-MS/MS analysis, linear peptides eluted with a single-peak XIC pattern, while the cross-linked peptide pairs with a transcyclized linker (consisting of a mixture of diastereomers) eluted with a two-peak XIC pattern in 68–100 % of cases. Cross-linked peptide pairs with the hydrolyzed linker were detected with a multi-peak XIC pattern consisting of two, three and four peaks in 77 %, 15 % and 8 % of cases, respectively. The four-peak XIC pattern corresponds to cross-linked peptide pairs with two positional isomers of the hydrolyzed linker containing a mixture of diastereomers. Tandem digestions increased the analytical value of the XICs by converting the large and hydrophobic cross-linked peptide pairs, that show a single-peak XIC pattern, to more hydrophilic species eluting in multi-peak XIC patterns. XICs are easily obtained in any LC-MS/MS analysis, contain valuable information about conjugation sites and side reaction detection, and are worthy of routine inclusion in bioconjugate characterization.

On the utility of the extracted ion chromatograms for assigning the conjugation sites and side reactions in bioconjugates synthesized by the maleimide-thiol chemistry