• electrospray ionization;
  • tandem mass spectrometry;
  • nonnatural amino acids;
  • hybrid peptides;
  • positional isomers


Two new series of Boc-N-α,δ-/δ,α- and β,δ-/δ,β-hybrid peptides containing repeats of L-Ala-δ5-Caa/δ5-Caa-L-Ala and β3-Caa-δ5-Caa/δ5-Caa-β3-Caa (L-Ala = L-alanine, Caa = C-linked carbo amino acid derived from D-xylose) have been differentiated by both positive and negative ion electrospray ionization (ESI) ion trap tandem mass spectrometry (MS/MS). MSn spectra of protonated isomeric peptides produce characteristic fragmentation involving the peptide backbone, the Boc-group, and the side chain. The dipeptide positional isomers are differentiated by the collision-induced dissociation (CID) of the protonated peptides. The loss of 2-methylprop-1-ene is more pronounced for Boc-NH-L-Ala-δ-Caa-OCH3 (1), whereas it is totally absent for its positional isomer Boc-NH-δ-Caa-L-Ala-OCH3 (7), instead it shows significant loss of t-butanol. On the other hand, second isomeric pair shows significant loss of t-butanol and loss of acetone for Boc-NH-δ-Caa-β-Caa-OCH3 (18), whereas these are insignificant for its positional isomer Boc-NH-β-Caa-δ-Caa-OCH3 (13). The tetra- and hexapeptide positional isomers also show significant differences in MS2 and MS3 CID spectra. It is observed that ‘b’ ions are abundant when oxazolone structures are formed through five-membered cyclic transition state and cyclization process for larger ‘b’ ions led to its insignificant abundance. However, b1+ ion is formed in case of δ,α-dipeptide that may have a six-membered substituted piperidone ion structure. Furthermore, ESI negative ion MS/MS has also been found to be useful for differentiating these isomeric peptide acids. Thus, the results of MS/MS of pairs of di-, tetra-, and hexapeptide positional isomers provide peptide sequencing information and distinguish the positional isomers. Copyright © 2010 John Wiley & Sons, Ltd.