Differentiation of isobaric cross-linked peptides prepared via maleimide
chemistry by 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. MALDI-MS and MS/MS are useful for
differentiating these isobaric species. METHODS: Four
cross-linked peptides with thiosuccinimide linkers were synthesized.
Analogs with the linker that were transformed into thiazine and/or the
hydrolyzed thiosuccinimide linkers were then generated by incubating the
samples at neutral or basic pH. All of the cross-linked peptides were
purified by rp-HPLC and differentiated by MALDI-MS, -MS/MS and UVPD.
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 gave 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 gave fragment ions resulting
from the cleavage of the newly formed amide bond in the linker that
arose from thiazine formation. CONCLUSIONS: The thiosuccinimide
(but not thiazine) form of the cross-linked peptide yielded individual
constituent peptides in MALDI-MS; MALDI-MS/MS showing specific
1,4-elimination for the thiosuccinimide form and cleavage at the newly
formed peptide bond via transcyclisation for the thiazine form.