The fragmentation characteristics of protonated alanylglycylglycine, [AGG+H]+, were investigated by tandem mass spectrometry in MALDI-TOF/TOF, ion trap, and hybrid sector instruments. b2 is the most abundant fragment ion in MALDI-TOF/TOF, ion trap, and hybrid sector metastable ion (MI) experiments, while y2 is slightly more abundant than b2 in collision activated dissociation (CAD) performed in the sector instrument. The A-G amide bond is cleaved on the a1-y2 pathway resulting in a proton-bound dimer of GG and MeCH=NH. Depending on the fragmentation conditions employed, this dimer can then (1) be detected as [AGG+H−CO]+, (2) dissociate to produce y2 ions, [GG+H]+, (3) dissociate to produce a1 ions, [MeCH=NH+H]+, or (4) rearrange to expel NH3 forming a [AGG+H−CO−NH3]+ ion. The activation method and the experimental timescale employed largely dictate which of, and to what extent, these processes occur. These effects are qualitatively rationalized with the help of quantum chemical and RRKM calculations. Two mechanisms for formation of the [AGG+H−CO−NH3]+ ion were evaluated through nitrogen-15 labeling experiments and quantum chemical calculations. A mechanism involving intermolecular nucleophilic attack and association of the GG and imine fragments followed by ammonia loss was found to be more energetically favorable than expulsion of ammonia in an SN2-type reaction.