Differentiation of three pairs of positional isomers of hybrid peptides with repeats of phenylalanine‐ β 3 ‐h‐valine/ β 3 ‐h‐valine‐phenylalanine by electrospray ionization tandem mass spectrometry

Electrospray ionization ion trap mass spectrometry has been used to distinguish three pairs of positional isomers of a new series of N‐blocked hybrid peptides derived from repeats of phenylalanine(D)‐ β 3 ‐h‐valine/ β 3 ‐h‐valine‐phenylalanine(D) (F β V/ β VF) non‐natural amino acids. MS n of protonated isomeric peptides produces characteristic fragmentation involving the peptide backbone, the Boc group and the side chain. F β V‐peptides can be distinguished from β VF‐peptides by the loss of R‐OH from [M+H−Boc+H] + , which is either of relatively low abundance or totally absent for the latter peptides. In contrast, β VF‐peptides show abundant Mannich base characteristic ions by the elimination of ammonia, and imine due to a retro‐Mannich cleavage. This fragmentation is absent for F β V‐peptides. When β ‐valine is at the C‐terminus, abundant b   n−1 +ions are produced. This is ascribed to the probable formation of a stable diketopiperazine structure, and this has been supported by the loss of H 2 O and CO in the CID spectra of b   n−1 +ions. The hybrid dipeptide acids have also been distinguished in negative ion mass spectrometry. Copyright © 2007 John Wiley & Sons, Ltd.