The fragmentation reactions of a number of a2 ions ([M−H−CO2]-) derived from dipeptides have been studied by energy-resolved mass spectrometry, isotopic labeling, and MS3 experiments. The general reaction sequence leading eventually to a deprotonated amine, is shown to occur, a reaction sequence first proposed by Styles and O'Hair (Rapid Commun. Mass Spectrom. 1998, 12, 809) from a study of the a2 ion derived from glycylglycine. When an amidic hydrogen (R2 = H) is present, the initial proton-transfer reaction 1 is nonreversible. However, when there is no amidic hydrogen, as in the a2 ions derived from H−Ala−Pro−OH or H−Gly−Sar−OH, the initial proton-transfer reaction 1 becomes reversible, leading to the interchange of N-bonded and C-bonded hydrogens. Ab initio calculations at the MP2/6-31+G(d) level of the energies and interconversion pathways of anions derived by deprotonation of glycine N-methylamide show a barrier of 8 kcal mol-1 for reaction 1, with reaction 2 being 23.8 kcal mol-1 endothermic. When an amidic hydrogen (R2 = H) is present, the amine-deprotonated species formed in reaction 1 abstracts a proton from the amide nitrogen to form the amide-deprotonated species, the most stable species on the potential energy surface. The system effectively becomes trapped in this low-energy well and exits upon activation by reactions 2 and 3 as observed when glycine N-methylamide is deprotonated directly. When no amidic hydrogen is present, this low-energy state does not exist, and reaction 1 becomes reversible, leading to the interchange of N-bonded and C-bonded hydrogens. In these cases, a significant population of the original a2 ion is formed, which fragments by the reaction