Differentiation between aspartic acid and iso-aspartic acid from asparagine deamidation has been previously reported using electron-involved dissociation (ExD) methods, such as electrontransfer dissociation (ETD) and electron-capture dissociation (ECD). c+57 and z-57 were confirmed to be a pair of diagnostic ions to identify iso-aspartic acid generated from either ETD or ECD. Asparagine deamidation first generates a succinimide intermediate prior to ringopening hydrolysis, which is analogous to thio-succinimide hydrolysis as shown in Figure . Figure shows the mechanism of asparagine deamidation and structures of diagnostic c-and zrelated ions. Figure shows the dissociation sites, and the structures of c-and z-related ions in both deamidation and thio-succinimide hydrolysis isomers. Figures and are the MS/MS spectra (m/z range: 100-2900) of [GEC+succinimide+H2O+R2+3H] 3+ precursor ions from peak 1 and 2, respectively, using EAD. The product ions in both figures show high agreement with each other. (I), (II) and (III) in Figures and are zoomed-in MS/MS spectra with different m/z range. Comparing Figures ) and 3B (I), we found a singly charged product ion with monoisotopic m/z of 365.0839, which is in accordance with [R1+Thio+57+H] + diagnostic ion with a -15 ppm mass difference. Similarly, by comparing Figures 3A (III) and 3B (III), we found a singly charged product ion with monoisotopic m/z of 1109.5403. This peak suggests the presence of [R2+Succ+H2O-57+H] + diagnostic ion with -12 ppm mass difference. Thus, we can confirm Figure represents the MS/MS spectrum of thio-Asp from peak 1, whereas Figure is the MS/MS spectrum of thio-isoAsp from peak 2. Observed m/z values of discussed ions all show similar extent of deviation to their theoretical values. A recalibration performed after acquisition corrects the m/z difference to within ±3 ppm (see Section S3 in the supporting information). Hence, the identification of ions of interest is accurate, despite the mass errors are close to the high end of tolerance.