Furthermore, we investigated the isomerism of the thiol-succinimide hydrolysis products. We successfully assigned structures to each isomeric product through an EAD MS/MS fragmentation without requiring reference materials using a previously reported approach. Using the diagnostic ions c+57, z-57 and z-12 for identifying thio-isoaspartic acid (thio-isoAsp) and z-44 for identifying thio-aspartic acid (thiol-Asp), we could confidently assign the structures to different isomer structures. Figure shows the relative abundances of thio-Asp and thio-isoAsp for hydrolyzed Linker-PL-containing tryptic peptides, GEC and SCDK, respectively. We observed a ~ 3:1 ratio between thio-isoAsp and thio-Asp from SCDK + 1 Hydrolyzed Linker-PL, as shown in Figure . However, Figure suggests a 1:1 ratio between two isomeric products on GEC + 1 Hydrolyzed Linker-PL structure. Geiger and Clarke reported a 3:1 ratio between iso-aspartic acid and aspartic acid resulted from asparagine deamidation. Since the thio-succinimide hydrolysis mechanism is structurally similar to asparagine deamidation, one could expect the ratio between two isomers from thio-succinimide hydrolysis should approach 3:1 as well. However, we observed different isomers ratios between Linker-PL conjugation sites on GEC and SCDK peptides. Thus, we hypothesize that the thio-succinimide hydrolysis isomerization might be impacted by the location of conjugation sites. We also noticed that the ratio between two isomers remains relatively unchanged for both GEC + 1 Hydrolyzed Linker-PL and SCDK + 1 Hydrolyzed Linker-PL across different timepoints.