A hydrolyzed N-propionylthiosuccinimide linker is cleaved by metastable fragmentation, increasing reliability of conjugation site identification in conjugate vaccines

IF 1.8 3区化学 Q4 BIOCHEMICAL RESEARCH METHODS

Rapid Communications in Mass Spectrometry Pub Date : 2024-07-21 DOI:10.1002/rcm.9859

Pablo E. Ramos-Bermúdez, Satomy Pousa, Paulo Carvalho, Rodrigo Soares Caldeira Brant, Michel Batista, Hironobu Hojo, Hilda E. Garay, Abel Roscoe, Alina Rodríguez Mallón, Vladimir Besada, Toshifumi Takao, Luis Javier González

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Abstract

RATIONALE

Conjugation sites are a quality attribute of conjugate vaccines. Proteolysis of bioconjugates synthesized by maleimide–thiol chemistry generates type 2 peptides with a hydrolyzed thiosuccinimide linker containing information on the conjugation sites. A mass spectrometry (MS)-cleavable linker could make the identification of conjugation sites by MS more reliable.

METHODS

Four synthetic type 2 peptides with a hydrolyzed thiosuccinimide linker were analyzed by matrix-assisted laser desorption ionization (MALDI) MS/MS with and without collision gas. These peptides were also partially labeled with ¹⁸O in the linker to confirm the proposed fragmentation mechanism. A conjugate vaccine with the hydrolyzed thiosuccinimide linker was reduced and S-alkylated, digested with trypsin and analyzed by liquid chromatography–MS/MS using collision-induced dissociation (CID) and higher-energy collisional dissociation (HCD) fragmentation methods at a normalized collision energy of 30.

RESULTS

A metastable fragmentation preferentially cleaves the newly formed pseudopeptide bond within the hydrolyzed thiosuccinimide linker of type 2 peptides to yield P + 71 and C + 98 ions. These ions make the assignment of conjugation sites more reliable. Partial ¹⁸O-labeling and MS/MS analysis confirmed the proposed structures. CID produces these ions as the two most intense signals more favorably than HCD. The latter also yields these ions, guarantees better sequence coverage and promotes other fragmentations in the linker.

CONCLUSIONS

Hydrolyzed thiosuccinimide linker is cleavable in MALDI and electrospray ionization MS/MS analysis by a gas-phase metastable fragmentation. The resulting fragment ions (P + 71 and C + 98) make the identification of conjugation sites more reliable. These results could be extended to self-hydrolyzing maleimides, which efficiently stabilize the thiosuccinimide linker upon hydrolysis, in antibody–drug conjugates.

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水解的 N-丙酰基硫代琥珀酰亚胺连接体可通过可转移碎片裂解，从而提高了共轭疫苗中共轭部位识别的可靠性。

理由共轭位点是共轭疫苗的一个质量属性。通过马来酰亚胺-硫醇化学合成的生物共轭物经蛋白水解后产生 2 型肽，水解后的硫代丁二酰亚胺连接体含有共轭位点信息。质谱（MS）可清除连接体可使质谱鉴定共轭位点更加可靠：方法：用基质辅助激光解吸电离（MALDI）质谱/质谱分析了带有水解硫代丁二酰亚胺连接体的四种合成 2 型肽，包括使用碰撞气体和不使用碰撞气体的情况。这些肽还在连接体中用 18O 进行了部分标记，以证实所提出的碎裂机制。水解硫代丁二酰亚胺连接体的共轭疫苗经还原和 S-烷基化后，用胰蛋白酶消化，并使用液相色谱-MS/MS，在归一化碰撞能量为 30 的条件下，采用碰撞诱导解离（CID）和高能碰撞解离（HCD）碎裂方法进行分析：结果：可转移的碎片优先裂解水解的硫代琥珀酰亚胺连接体中新形成的假肽键，产生 P + 71 和 C + 98 离子。这些离子使共轭位点的确定更加可靠。部分 18O 标记和 MS/MS 分析证实了所提出的结构。与 HCD 相比，CID 产生的这些离子是两个最强烈的信号。后者也能产生这些离子，保证了更好的序列覆盖率，并促进了连接体中的其他碎片：结论：在 MALDI 和电喷雾电离 MS/MS 分析中，水解的硫代丁二酰亚胺连接体可通过气相易变碎片裂解。由此产生的碎片离子（P + 71 和 C + 98）使共轭位点的鉴定更加可靠。这些结果可以推广到抗体-药物共轭物中的自水解马来酰亚胺中，水解后的马来酰亚胺能有效稳定硫代丁二酰亚胺连接体。

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来源期刊

Rapid Communications in Mass Spectrometry 化学-分析化学

CiteScore

4.10

自引率

5.00%

发文量

219

审稿时长

2.6 months

期刊介绍： Rapid Communications in Mass Spectrometry is a journal whose aim is the rapid publication of original research results and ideas on all aspects of the science of gas-phase ions; it covers all the associated scientific disciplines. There is no formal limit on paper length ("rapid" is not synonymous with "brief"), but papers should be of a length that is commensurate with the importance and complexity of the results being reported. Contributions may be theoretical or practical in nature; they may deal with methods, techniques and applications, or with the interpretation of results; they may cover any area in science that depends directly on measurements made upon gaseous ions or that is associated with such measurements.