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Differentiation of isobaric cross-linked peptides prepared via maleimide chemistry by MALDI-MS and MS/MS.
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  • Toshifumi Takao,
  • Luis Gonzalez,
  • Satomy Pousa,
  • Hironobu Hojo,
  • Shio Watanabe,
  • Daisuke Higo,
  • Alina Rodriguez Mallon
Toshifumi Takao
Osaka Daigaku Tanpakushitsu Kenkyujo

Corresponding Author:tak@protein.osaka-u.ac.jp

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Luis Gonzalez
Centro de Ingenieria Genetica y Biotecnologia
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Satomy Pousa
Centro de Ingenieria Genetica y Biotecnologia
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Hironobu Hojo
Osaka Daigaku Tanpakushitsu Kenkyujo
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Shio Watanabe
Thermo Fisher Scientific Inc Mitato-ku Tokyo
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Daisuke Higo
Thermo Fisher Scientific Inc Mitato-ku Tokyo
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Alina Rodriguez Mallon
Centro de Ingenieria Genetica y Biotecnologia
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Abstract

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.
21 Aug 2023Submitted to Rapid Communications in Mass Spectrometry
21 Aug 2023Submission Checks Completed
21 Aug 2023Assigned to Editor
21 Aug 2023Review(s) Completed, Editorial Evaluation Pending
25 Aug 2023Reviewer(s) Assigned
25 Sep 2023Editorial Decision: Revise Minor
01 Oct 20231st Revision Received
01 Oct 2023Review(s) Completed, Editorial Evaluation Pending
01 Oct 2023Submission Checks Completed
01 Oct 2023Assigned to Editor
01 Oct 2023Editorial Decision: Accept