N-propionyl thiosuccinimide linker upon hydrolysis becomes into a cleavable crosslinker by a gas phase metastable fragmentation and makes more reliable the conjugation sites identification.

Maleimide thiol chemistry is widely used for the synthesis of conjugate vaccines and antibody-drug-conjugates. Proteolysis of a conjugate vaccine mainly generates linear and type 2 peptides (cross-linked peptides) with a thiosuccinimide linker and its stabilized forms (thiazine, and/or hydrolyzed thiosuccinimide). Type 2 peptides contain valuable information on the conjugation sites. Cleavable crosslinkers could make more reliable the conjugation sites identifications, but based on a different chemistry, they are used to study protein-protein interactions and not in the synthesis of bioconjugates. Hydrolysis not only stabilizes the thiosuccinimide linker, but also turns it into a cleavable crosslinker via a gas phase metastable fragmentation observed in MALDI- and ESI-MS/MS analysis. The carboxyl group in the hydrolyzed thiosuccinimide linker provides a mobile proton that destabilizes the nearest pseudopeptide bond and, through the rearrangement of a five-membered ring intermediate, yields two intense linker fragment ions named here as P+71 and C+98. Specific 18O-labeling of the hydrolyzed thiosuccinimide linker confirms that P+71 ion, has the epsilon amino group of Lys modified by a -alanine residue while C+98 ion has the Cys residue S-alkylated by a succinic anhydride molecule. P+71 and C+98 provide information on the molecular masses of the crosslinked peptides pair, decrease the number of unassigned signals in the MS/MS spectra; and their backbone fragment ions increased the sequence coverage and make more reliable the assignment of conjugation sites. Stepped HCD is the fragmentation method of choice because in a single MS/MS spectrum several linker fragment ions and bn/yn ions were observed.