The following peptides have been examined in this study: GLDFG(OH), caeridin 1.1 [GLLDGLLGLGGL(NH2)], 11 Ala citropin 1.1 [GLFDVIKKVAAVIGGL(NH2)], Crinia angiotensin [APGDRIYVHPF(OH)] and their isoAsp isomers. It is not possible to differentiate between Asp- and isoAsp-containing peptides (used in this study) using negative ion electrospray mass spectrometry. This is because the isoAsp residue cleaves to give the same fragment anions as those formed by δ and γ backbone cleavage of Asp. The isoAsp fragmentations are as follows: RNHCH(CO2H)−CHCONHR′ → [RNH−(HO2CCHCHCONHR′)] → RNH−+HO2CCHCHCONHR′ and RNHCH(CO2H)−CHCONHR′ → [RNH−(HO2CCHCHCONHR′] → −O2CCHCHCONHR′+RNH2. Calculations at the HF/6-31+G(d)//AM1 level of theory indicate that the first of these isoAsp cleavage processes is endothermic (by +115 kJ mol−1), while the second is exothermic (−85 kJ mol−1). The barrier to the highest transition state is 42 kJ mol−1. No diagnostic cleavage cations were observed in the electrospray mass spectra of the MH+ ion of the Asp- and isoAsp-containing peptides (used in this study) to allow differentiation between these two amino acid residues. Copyright © 2009 John Wiley & Sons, Ltd.