Another notable difference is [R2+Succ+H2O-44+2H] 2+ (indicated by pink arrow) shows significantly higher intensity in Figure , whereas the intensity of the same product ion in Figure is low. Figures and show zoomed-in MS/MS spectra from both peak 1 and 2 at m/z 555-570 range, respectively. The higher signal intensity of [R2+ Succ+H2O-44+2H] 2+ in Figure suggests the CO2 loss after forming [R2+ Succ+H2O+2H] 2+ ion of thio-Asp is significantly enhanced. This finding aligns with the ExD dissociation results of aspartic acid in deamidation. We also observed a doubly-charged product ion with a monoisotopic m/z of 561.2728 with enhanced intensity in Figure (II), but low intensity in Figure (II). We propose the structure of thio+succinimide+H2O+R2-CO2 (Figure ). We denote this product ion as [R2+ThioSucc+H2O-44+2H] 2+ . Although both thio-Asp and thio-isoAsp are capable of CO2 loss after forming the [R2+ThioSucc+H2O+2H] 2+ product ion, clearly, thio-isoAsp is more prone to this neutral loss. We also found the [R2+ThioSucc+H2O+2H] 2+ product ion in both thio-Asp and thio-isoAsp with similar intensities, as shown in Figure and Figure . Thus, [R2+ThioSucc+H2O-44+2H] 2+ product ion can serve as an additional diagnostic ion for differentiating thio-Asp and thio-isoAsp. To our knowledge, this product ion has never been reported in the context of differentiation of deamidation isomers. Hence, this diagnostic ion is distinctive of the thio-succinimide system. We infer that the generation of this unique product ion is related to the thiol group linked with the succinimide ring and R2.