{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,3,8]],"date-time":"2026-03-08T16:02:17Z","timestamp":1772985737024,"version":"3.50.1"},"reference-count":29,"publisher":"Wiley","issue":"23","license":[{"start":{"date-parts":[[2024,9,17]],"date-time":"2024-09-17T00:00:00Z","timestamp":1726531200000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"},{"start":{"date-parts":[[2024,9,17]],"date-time":"2024-09-17T00:00:00Z","timestamp":1726531200000},"content-version":"tdm","delay-in-days":0,"URL":"http:\/\/doi.wiley.com\/10.1002\/tdm_license_1.1"}],"funder":[{"DOI":"10.13039\/100004325","name":"AstraZeneca","doi-asserted-by":"publisher","id":[{"id":"10.13039\/100004325","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["analyticalsciencejournals.onlinelibrary.wiley.com"],"crossmark-restriction":true},"short-container-title":["Rapid Comm Mass Spectrometry"],"published-print":{"date-parts":[[2024,12,15]]},"abstract":"<jats:sec>\n                    <jats:title>Rationale<\/jats:title>\n                    <jats:p>Isomerism can be an important aspect in pharmaceutical drug development. Identification of isomers can provide insights into drug pharmacology and contribute to better design of drug molecules. The general approaches to differentiate isomers include Fourier\u2010transform infrared spectroscopy (FTIR), nuclear magnetic resonance (NMR), and circular dichroism. Additionally, a commonly used method to differentiate isomers is liquid chromatography coupled with mass spectrometry (LC\u2010MS). Notably, LC\u2010MS is routinely applied to leucine and isoleucine differentiation to facilitate protein sequencing. This work focuses on isomer differentiation of widely employed thio\u2010succinimide structure bridging the antibody backbone and linker\u2010payload of antibody\u2010drug conjugates (ADCs). Thio\u2010succinimide hydrolysis stabilizes the payload\u2010protein structure while generating a pair of constitutional isomers: thio\u2010aspartyl and thio\u2010isoaspartyl.<\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Methods<\/jats:title>\n                    <jats:p>This paper introduces a hybrid method using ligand binding assay (LBA) and liquid chromatography coupled with tandem MS (LC\u2013MS\/MS) to reveal isomerization details of thio\u2010succinimide hydrolysis over time in plasma samples incubated with ADC. Application of two orthogonal dissociation methods, collision\u2010induced dissociation (CID) and electron\u2010activated dissociation (EAD) revealed different MS\/MS spectra for this pair of isomers. This observation enables a unique approach in distinguishing thio\u2010succinimide hydrolysis isomers.<\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Results<\/jats:title>\n                    <jats:p>\n                      We observed signature [R\n                      <jats:sub>1<\/jats:sub>\n                      \u00a0+\u2009Thio\u2009+\u200957\u2009+\u2009H]\n                      <jats:sup>+<\/jats:sup>\n                      , [R\n                      <jats:sub>2<\/jats:sub>\n                      \u00a0+\u2009Succ\u2009+\u2009H\n                      <jats:sub>2<\/jats:sub>\n                      O\u2009\u2212\u200957\u2009+\u2009H]\n                      <jats:sup>+<\/jats:sup>\n                      , and [R\n                      <jats:sub>2<\/jats:sub>\n                      \u00a0+\u2009Succ\u2009+\u2009H\n                      <jats:sub>2<\/jats:sub>\n                      O\u2009\u2212\u200944\u2009+\u20092H]\n                      <jats:sup>2+<\/jats:sup>\n                      product ions (Succ = succinimide) that differentiated thio\u2010aspartyl and thio\u2010isoaspartyl isomers using EAD. A newly discovered [R\n                      <jats:sub>2<\/jats:sub>\n                      \u00a0+\u2009ThioSucc\u2009+\u2009H\n                      <jats:sub>2<\/jats:sub>\n                      O\u2009\u2212\u200944\u2009+\u20092H]\n                      <jats:sup>2+<\/jats:sup>\n                      ion also served as additional evidence that further supported our findings.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Conclusions<\/jats:title>\n                    <jats:p>This study is a first\u2010to\u2010date identification of thio\u2010succinimide hydrolysis isomers without using synthesized reference materials. This approach should be applicable to all thio\u2010succinimide\u2010linked molecules. Correct identification of thio\u2010succinimide hydrolysis isomers may eventually benefit the development of ADCs in the future.<\/jats:p>\n                  <\/jats:sec>","DOI":"10.1002\/rcm.9910","type":"journal-article","created":{"date-parts":[[2024,9,17]],"date-time":"2024-09-17T14:23:54Z","timestamp":1726583034000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":4,"title":["Reference\u2010free thio\u2010succinimide isomerization characterization by electron\u2010activated dissociation"],"prefix":"10.1002","volume":"38","author":[{"given":"Junyan","family":"Yang","sequence":"first","affiliation":[{"name":"Integrated Bioanalysis, Clinical Pharmacology &amp; Safety Sciences, R&amp;D AstraZeneca  South San Francisco California USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Jiaqi","family":"Yuan","sequence":"additional","affiliation":[{"name":"Integrated Bioanalysis, Clinical Pharmacology &amp; Safety Sciences, R&amp;D AstraZeneca  South San Francisco California USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Yue","family":"Huang","sequence":"additional","affiliation":[{"name":"Integrated Bioanalysis, Clinical Pharmacology &amp; 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