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

Maleimide-thiol chemistry is widely used to synthesize antibody-drug conjugates and conjugate vaccines. The set of MS/MS spectra automatically assigned to proteolytic type 2 peptides with a thiosuccinimide linker and its stabilized products (thiazine and hydrolyzed species), contains valuable information on the conjugation sites. Sample processing before LC-MS/MS analysis transforms the thiosuccinimide linker into its stabilized products. The MS/MS spectra of type 2 peptides must be validated based on objective criteria. The extracted ion chromatogram (XIC) has not been considered previously for this purpose. In the LC-MS/MS analysis, linear peptides eluted in a one-fraction XIC as expected. On the contrary, depending on the analyzed proteolytic digestion, the type 2 peptides with a thiazine linker were detected in 77-100 % of the cases with a two-fraction XIC pattern, probably composed by a mixture of diastereomers. Type 2 peptides with the hydrolyzed thiosuccinimide linker were detected with a mixed-pattern XIC in two, three and four elution fractions in 77 %, 15 % and 8 % of the cases, respectively. The four-fraction XIC pattern is probably composed by the full-chromatographic separation of type 2 peptides cross-linked by two positional isomers of the thiosuccinamic acid linker containing a chiral carbon. Tandem digestions increased the number of type 2 peptides detected with a multiple-fractions XIC pattern. XIC of proteolytic peptides is a relevant source of information considered for data validation. Transcyclization and hydrolysis not only stabilize the thiosuccinimide linker but also permit, together with XIC, a more reliable identification of the conjugation sites by LC-MS/MS analysis.