Isotope labeling and theoretical study of the formation of a3* ions from protonated tetraglycine

Extensive C-13, N-15, and H-2 labeling of tetraglycine was used to investigate the b(3)(+) -> a(3)(*) reaction during low-energy collision-induced dissociation (CID) in a quadrupole ion-trap mass spectrometer. The patterns observed with respect to the retention or elimination of the isotope labels demonstrate that the reaction pathway involves elimination of CO and NH3. The ammonia molecule includes 2 H atoms from amide or amino positions, and one from an a-carbon position. The loss of NH3 does not involve elimination of the N-terminal amino group but, instead, the N atom of the presumed oxazolone ring in the b(3)(+) ion. The CO molecule eliminated is the carbonyl group of the same oxazolone ring, and the a-carbon H atom is transferred from the amino acid adjacent to the oxazolone ring. Quantum chemical calculations indicate a multistep reaction cascade involving CO loss on the b(3) -> a(3) pathway and loss of NH=CH2 from the a(3) ion to form b(2). In the postreaction complex of b(2) and NH=CH2, the latter can be attacked by the N-terminal amino group of the former. The product of this attack, an isomerized a(3) ion, can eliminate NH3 from its N-terminus to form a(3)(*). Calculations suggest that the ammonia and a(3)(*) species can form various ion-molecule complexes, and NH3 can initiate relay-type mobilization of the oxazolone H atoms from alpha-carbon positions to form a new oxazolone isomer. This multiple-step reaction scheme clearly explains the isotope labeling results, including unexpected scrambling of H atoms from alpha-carbon positions.