Neutral loss of water from the amide bond induced by the His side chain has been reported. The proposed fragmentation pathway is a retro-Ritter reaction catalyzed by the imidazole nitrogen. In our MS/MS study of the neuropeptide GAHKNYLRFamide, we observed that the neutral loss of water from the b(3) ion is abundant. The b(3) ion has a His residue at the C-terminus. As reported previously, in the b ions with His at the C-terminus, the imidazole residue is connected to the carbonyl carbon to form a five-membered ring. Therefore, it is unlikely that the neutral loss of water from the b(3) ion is catalyzed by the imidazole nitrogen. Through MS2 and MS3 studies of a synthetic peptide standard AGHKLL and its chemically labeled and isotope-encoded forms, we discovered that the water loss from the b(3) ion involves the carbonyl group of His, the hydrogen connected to the alpha-carbon of Gly, and the amide hydrogen of His. We also discovered the formation of an unusual c(x) ion in peptides with a Lys or Arg residue at the (x + 1) position of the peptide.