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The following peptides have been examined in this study: GLDFG(OH), caeridin 1.1 [GLLDGLLGLGGL(NH(2))], 11 Ala citropin 1.1 [GLFDVIKKVAAVIGGL(NH(2))], 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 delta and gamma backbone cleavage of Asp. The isoAsp fragmentations are as follows: RNHCH(CO(2)H)(-)CHCONHR' --> [RNH(-)(HO(2)CCH=CHCONHR')] --> RNH(-)+HO(2)CCH=CHCONHR' and RNHCH(CO(2)H)(-)CHCONHR' --> [RNH(-)(HO(2)CCH=CHCONHR'] --> (-)O(2)CCH=CHCONHR'+RNH(2). 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 (c) 2009 John Wiley & Sons, Ltd.)

[M-H](-) anions from small diphosphopeptides (phosphate groups on Ser, Thr or Tyr) show characteristic peaks corresponding to m/z 177 (H(3)P(2)O(7) (-)), 159 (HP(2)O(6) (-)) and sometimes [(M-H)(-)-H(4)P(2)O(7)](-). M/z 177 and m/z 159 are major peaks in the spectra of small peptides with 1,2, 1,3, 1,4, 1,5 and 1,6 diphosphate substitution, which means that the decomposing [M-H](-) anions must have flexible structures in order for the two phosphate groups to interact with each other. Peptides where the two phosphate groups are more than six amino acid residues apart have not been studied. Theoretical calculations indicate that m/z 177 is formed in a strongly exothermic reaction involving facile nucleophilic interaction between the two phosphate groups: m/z 159 is formed by loss of water from energised m/z 177.