Differentiation of isobaric cross‐linked peptides prepared via maleimide chemistry using MALDI‐MS and MS/MS

Rationale The thiosuccinimide linker is widely used in the synthesis of bioconjugates. However, it is susceptible to hydrolysis and is transformed into its hydrolyzed and/or the isobaric thiazine forms, the latter of which is a fairly common product in a conjugate that contains a cysteinyl peptide. Matrix‐assisted laser desorption/ionization‐mass spectrometry (MALDI‐MS) and matrix‐assisted laser desorption/ionization‐tandem mass spectrometry (MALDI‐MS/MS) are useful for differentiating these isobaric species. Methods Four cross‐linked peptides with thiosuccinimide linkers were synthesized. Analogs with linkers that were transformed into thiazine and/or the hydrolyzed thiosuccinimide linkers were then synthesized by incubating the samples at neutral or basic pH. All the cross‐linked peptides were purified using RP‐HPLC (reversed‐phase high‐performance liquid chromatography) and differentiated using MALDI‐MS, MALDI‐MS/MS, and ultraviolet photodissociation. Results A cysteinyl peptide‐containing conjugate, the thiosuccinimide form, was largely transformed into the hydrolyzed or thiazine forms after incubation at neutral or basic pH. MALDI‐MS allowed the three forms to be differentiated: the thiosuccinimide and its hydrolysis product yielded two constituent peptides after reductive cleavage between the Cys and succinimide moieties; no fragment ions were produced from the thiazine form. In addition, MALDI‐MS/MS of the thiosuccinimide form yielded two pairs of complementary fragment ions via 1,4‐elimination: Cys‐SH and maleimide, and dehydro‐alanine and thiosuccinimide, which are different from those produced via reductive cleavage in MALDI‐MS. The thiazine form yielded fragment ions resulting from the cleavage of the newly formed amide bond in the linker that resulted from thiazine formation. Conclusions The thiosuccinimide (but not thiazine) form of the cross‐linked peptide yielded individual constituent peptides using MALDI‐MS and MALDI‐MS/MS, showing specific 1,4‐elimination for the thiosuccinimide form and cleavage at the newly formed peptide bond via transcyclization for the thiazine form.