journal article

Journal Article

Text

Rapid Communications in Mass Spectrometry. 2024, 38(2), p. e9660

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

John Wiley and Sons Ltd

English

Text

2024-01-30

https://doi.org/10.1002/rcm.9660

This is the peer reviewed version of the following article: González L.J., Pousa S., Hojo H., et al. Differentiation of isobaric cross-linked peptides prepared via maleimide chemistry using MALDI-MS and MS/MS. Rapid Communications in Mass Spectrometry 38, e9660 (2024), which has been published in final form at https://doi.org/10.1002/rcm.9660. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Self-Archiving. This article may not be enhanced, enriched or otherwise transformed into a derivative work, without express permission from Wiley or by statutory rights under applicable legislation. Copyright notices must not be removed, obscured or modified. The article must be linked to Wiley’s version of record on Wiley Online Library and any embedding, framing or otherwise making available the article or pages thereof by third parties from platforms, services and websites other than Wiley Online Library must be prohibited.

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.

© 2023 John Wiley & Sons Ltd.

94635

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

John Wiley and Sons Ltd

English

2024-01-30

Rapid Communications in Mass Spectrometry

38

2

e9660

https://doi.org/10.1002/rcm.9660

https://hdl.handle.net/11094/94635