Mass Spectrometry Laboratory, Department of Proteomics, Center for Genetic Engineering and Biotechnology, Havana, Cuba.
Laboratory of Protein Organic Chemistry, Institute for Protein Research, Osaka University, Osaka, Japan.
Rapid Commun Mass Spectrom. 2024 Jan 30;38(2):e9660. doi: 10.1002/rcm.9660.
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.
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.
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.
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.
硫代琥珀酰亚胺连接子广泛用于生物缀合物的合成。然而，它易水解，并转化为其水解和/或等排噻嗪形式，后者是含有半胱氨酸肽的缀合物中相当常见的产物。基质辅助激光解吸/电离质谱（MALDI-MS）和基质辅助激光解吸/电离串联质谱（MALDI-MS/MS）可用于区分这些等排体。
合成了四个带有硫代琥珀酰亚胺连接子的交联肽。然后通过在中性或碱性 pH 下孵育样品，合成了连接子转化为噻嗪和/或水解的硫代琥珀酰亚胺连接子的类似物。所有交联肽均通过反相高效液相色谱（RP-HPLC）纯化，并通过 MALDI-MS、MALDI-MS/MS 和紫外光解离进行区分。
在中性或碱性 pH 孵育后，含有半胱氨酸肽的缀合物的硫代琥珀酰亚胺形式主要转化为水解或噻嗪形式。MALDI-MS 允许区分这三种形式：Cys 和琥珀酰亚胺部分之间的还原裂解后，硫代琥珀酰亚胺及其水解产物产生两个组成肽；噻嗪形式不产生片段离子。此外，硫代琥珀酰亚胺形式的 MALDI-MS/MS 通过 1,4-消除产生两对互补的片段离子：Cys-SH 和马来酰亚胺，以及脱氢丙氨酸和硫代琥珀酰亚胺，这与 MALDI-MS 中通过还原裂解产生的不同。噻嗪形式产生的片段离子来自于通过噻嗪形成形成的连接子中新形成的酰胺键的裂解。
交联肽的硫代琥珀酰亚胺（而非噻嗪）形式通过 MALDI-MS 和 MALDI-MS/MS 产生单个组成肽，表明硫代琥珀酰亚胺形式的特异性 1,4-消除和噻嗪形式的通过反环化在新形成的肽键上的裂解。
