The glutamine amidotransferase (GATase) from Methanocaldococcus jannaschii (Mj) harbours a stable post-translational modification, succinimide (SNN). As shown from earlier studies, the spontaneous deamidation of Asn109 leading to the formation of stable SNN in MjGATase, imparts hyper-thermostability to the protein. To examine which of the residues in the environment of Asn109 catalyses the conversion of Asn to SNN, several mutants of MjGATase were generated. The intact protein masses of these mutants along with the wild-type enzyme were obtained through mass spectrometric analysis. The wild-type enzyme shows a mass difference of 17 Da due to the formation of SNN whereas, the MjGATase mutants harboring unmodified Asn109 would retain the mass corresponding to that obtained from the sequence. The mass analysis of 12 MjGATase mutant proteins showed varied levels retaining intact Asn109 residue. Two double mutants showed almost entire population with the expected mass, thus, highlighting the involvement of the neighbouring residues in formation of SNN.