A stable post-translational modification, succinimide (SNN), found in Methanocaldococcus jannaschii (Mj) glutamine amidotransferase (GATase) is shown to impart hyper-thermostability to the protein. To explore the role of neighbouring residues in enabling deamidation of Asn109 to SNN, intact protein mass analysis on 12 mutants of MjGATase was performed. Among these mutants, 8 showed varied proportions of mass corresponding to unmodified Asn109. SNN is known to undergo rapid hydrolysis to Asp/iso-Asp, hence, this population can also exist. As Asn intact and Asp/iso-Asp populations differ by only 1 Da, ambiguity exists in their assignment. Therefore, to determine the true levels of unmodified Asn109 population, in-gel trypsin digestion followed by MS/MS analysis of the tryptic peptides for 8 mutants and wild-type of MjGATase was conducted. The MS/MS analysis of two double mutants assured that they indeed retain Asn109 intact, forming a major population. This study points toward the presence of internal catalysts that enable the deamidation of Asn109 to SNN in MjGATase.