We observed similar findings for the THT GSH adduct. We discovered THTC-GSH fragment ion in the CID spectra of THT GSH adduct from peak 4, as shown in Figure . Thus, peak 4 corresponds to the GSH adduct at the first Linker-PL conjugation site, whereas peaks 1, 2 and 3 correspond to GSH adduct at the second Linker-PL conjugation site. By comparing the AUC of each peak in the XICs for both THT Cys and GSH adduct, we found endogenous Cys shows strong preference for forming adduct at the first linker-payload conjugation site on THT peptide of Hc, as indicated in Figure . However, GSH does not show a clear site preference on forming GSH adduct. Summing the AUCs of all THT Cys and GSH adducts together, we discovered that the first Linker-PL conjugation site shows a higher possibility of forming thiol group adduct (Figure ).