The thio-succinimide hydrolysis product is not susceptible to linker-payload deconjugation, and our bottom-up data demonstrated that linker-payload ring opening hydrolysis is the dominant reaction consistent with our previous observations using LC-HRMS explaining AZD8205 stability. However, a very minor pool of AZD8205 undergoes deconjugation of the linkerpayload. This biotransformation reaction then leads to cysteine and GSH adducts by disulfide bond formation with the liberated thiol. Using a LC-MRM approach, we confirmed the presence of Cys and GSH adducts on the Hc of AZD8205. Figures show the XICs and mass spectrum of THT peptide Cys adduct. This biotransformed species comes from one linker-payload deconjugation followed by Cys adduct, whereas the other conjugated linker-payload undergoes hydrolysis. THT Cys adduct is not detected at 0 hr, and its concentration increases with longer incubation time, as shown in Figure . The mass spectrum in Figure confirms the identity of THT Cys adduct at MS level. At 168 hr, there are mainly three peaks (labelled as 1, 2 and 3) observed in the XIC of THT Cys adduct biotransformation species. We hypothesize they might originate from the deconjugation of both linker-payloads in the THT peptide that subsequently led to the formation of the Cys adduct, while the other Linker-PL hydrolysis generates two constitutional isomers discussed previously. Figures and show CID and EAD MS/MS spectra of THT Cys adduct product (RT: 53.4 -55.2 minute). CID MS/MS spectrum shows multiple b and y ions matching THT peptide and linker-payload fragments. In particular, a THTC-Cys fragment is observed in Figure , validating our hypothesis of biotransformation of linker-payload deconjugation followed by cysteinylation. Furthermore, EAD MS/MS spectrum provides additional evidence on THT Cys adduct formation with a unique peak representing THT + 1 Hydrolyzed Linker-PL being displayed in Figure .