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
{"title":"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。","authors":"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","doi":"10.1002/rcm.9859","DOIUrl":null,"url":null,"abstract":"<div>\n \n \n <section>\n \n <h3> RATIONALE</h3>\n \n <p>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.</p>\n </section>\n \n <section>\n \n <h3> METHODS</h3>\n \n <p>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 <sup>18</sup>O in the linker to confirm the proposed fragmentation mechanism. A conjugate vaccine with the hydrolyzed thiosuccinimide linker was reduced and <i>S</i>-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.</p>\n </section>\n \n <section>\n \n <h3> RESULTS</h3>\n \n <p>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 <sup>18</sup>O-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.</p>\n </section>\n \n <section>\n \n <h3> CONCLUSIONS</h3>\n \n <p>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.</p>\n </section>\n </div>","PeriodicalId":225,"journal":{"name":"Rapid Communications in Mass Spectrometry","volume":"38 18","pages":""},"PeriodicalIF":1.8000,"publicationDate":"2024-07-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":"{\"title\":\"A hydrolyzed N-propionylthiosuccinimide linker is cleaved by metastable fragmentation, increasing reliability of conjugation site identification in conjugate vaccines\",\"authors\":\"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\",\"doi\":\"10.1002/rcm.9859\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<div>\\n \\n \\n <section>\\n \\n <h3> RATIONALE</h3>\\n \\n <p>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.</p>\\n </section>\\n \\n <section>\\n \\n <h3> METHODS</h3>\\n \\n <p>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 <sup>18</sup>O in the linker to confirm the proposed fragmentation mechanism. A conjugate vaccine with the hydrolyzed thiosuccinimide linker was reduced and <i>S</i>-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.</p>\\n </section>\\n \\n <section>\\n \\n <h3> RESULTS</h3>\\n \\n <p>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 <sup>18</sup>O-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.</p>\\n </section>\\n \\n <section>\\n \\n <h3> CONCLUSIONS</h3>\\n \\n <p>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.</p>\\n </section>\\n </div>\",\"PeriodicalId\":225,\"journal\":{\"name\":\"Rapid Communications in Mass Spectrometry\",\"volume\":\"38 18\",\"pages\":\"\"},\"PeriodicalIF\":1.8000,\"publicationDate\":\"2024-07-21\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Rapid Communications in Mass Spectrometry\",\"FirstCategoryId\":\"92\",\"ListUrlMain\":\"https://onlinelibrary.wiley.com/doi/10.1002/rcm.9859\",\"RegionNum\":3,\"RegionCategory\":\"化学\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q4\",\"JCRName\":\"BIOCHEMICAL RESEARCH METHODS\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Rapid Communications in Mass Spectrometry","FirstCategoryId":"92","ListUrlMain":"https://onlinelibrary.wiley.com/doi/10.1002/rcm.9859","RegionNum":3,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q4","JCRName":"BIOCHEMICAL RESEARCH METHODS","Score":null,"Total":0}
A hydrolyzed N-propionylthiosuccinimide linker is cleaved by metastable fragmentation, increasing reliability of conjugation site identification in conjugate vaccines
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.
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.
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