Thermodynamics and Mechanisms of Protonated Asparaginyl-Glycine Decomposition

Deamidation at asparagine residues, a spontaneous post-translational modification in proteins, plays a significant role in various biological processes and degenerative diseases. In the current work, we present a full description of the deamidation process as well as other key fragmentations (dehydration, peptide bond cleavage, and loss of 2 NH₃) from protonated asparaginyl-glycine, H⁺(AsnGly), by studying its kinetic energy dependent collision-induced dissociation (CID) with Xe using a guided ion beam tandem mass spectrometer. These results are compared with those for sustained off-resonance irradiation (SORI)-CID of H⁺(AsnGly) with Ar in a Fourier transform ion cyclotron resonance mass spectrometer. Computationally, simulating annealing methodology and a series of relaxed potential energy scans at the B3LYP/6-31G(d) level were performed to identify all intermediate and transition state (TS) structures for each key reaction. All species were further optimized at the B3LYP and B3LYP-GD3BJ/6-311+G(d,p) levels of theory. Single point energies of all major reaction species were calculated at the B3LYP, B3P86, MP2(full), and B3LYP-GD3BJ levels of theory and using M06-2X for rate-limiting species. Relative energies of intermediates, TSs, and products allow characterization of the elementary and rate limiting steps in H⁺(AsnGly) decomposition. By combining experimental and computational results, the complete mechanistic nature of H⁺(AsnGly) deamidation and other fragmentations is explored and compared to the previously studied H⁺(Asn) complex. The influence of water solvation on key TSs is also explored. On a fundamental level, this analysis will aid in understanding the thermodynamic and kinetic characteristics of the key intramolecular interactions involved in deamidation, dehydration, and other important fragmentations of peptides.