Neutral losses are not limited to the product ions generated from ExD dissociation. [M-60+H] + was reported to be a diagnostic ion for Asp in deamidation. Figures and show the zoomed-in MS/MS spectra of Figures and at m/z 1410-1440. To our surprise, the [M-60+H] + product ions (observed monoisotopic m/z 1414.6355, 10 ppm difference to its theoretical value) in both Figures and show similar intensity. Thus, [M-60+H] + cannot differentiate thio-Asp and thio-isoAsp, and we think the 60 Da neutral loss might come from the contribution of R2. We also observed peaks with monoisotopic m/z 1430.6428, (-7 ppm difference to theoretical m/z of [M-44+H] + ) in both isomers. The similar intensities of [M-44+H] + and [M-60+H] + suggest that neutral losses to the whole precursor ion cannot distinguish thio-Asp and thio-isoAsp. Furthermore, we also investigated another tryptic peptide SCDK that contains succinimide+R2 from ADC1 followed by the same sample preparation steps. The SCDK+succinimide+R2 is mostly hydrolyzed after 168 h of incubation, as shown in Figure . Both XICs of nonhydrolyzed and hydrolyzed at 0 and 168 h in Figures and support the same conclusion. Additionally, Figure shows two distinct peaks that were well separated under current LC gradient. The presence of two peaks suggests the formation of isomers after thio-succinimide hydrolysis. This finding aligns with our observations of GEC-linked succinimide structure.