Electrospray ionization tandem mass spectrometry of protonated and alkali-cationized Boc-N-protected hybrid peptides containing repeats of D-Ala-APyC and APyC-D-Ala: Formation of [b_n–1 + OCH₃ + Na]⁺ and [b_n–1 + OH + Na]⁺ ions

RATIONALE

Differentiation and structural characterization of positional isomers of non-natural amino acid hybrid peptides by using electrospray ionization tandem mass spectrometry (ESI-MSⁿ) is desirable because of their fundamental importance from the view point of peptide mass spectrometry and also of their increasing importance in the area of research towards biomedical and material applications; hence, the present study is undertaken.

METHODS

Electrospray ionization ion-trap tandem mass spectrometry (ESI-MSⁿ) was used to characterize and differentiate three pairs of positional isomers of Boc-N-protected hybrid peptides containing repeats of D-Ala-APyC and APyC-D-Ala (D-Ala = D-alanine and APyC = trans-3-aminopyran-2-carboxylic acid).

RESULTS

ESI-MSⁿ spectra of protonated and alkali-cationized positional isomeric peptides display characteristic fragmentation involving the peptide backbone, the Boc group, and the side chain. It is observed that abundant rearrangement ions [b_n–1 + OCH₃ + Na]⁺ or [b_n–1 + OH + Na]⁺ are formed when D-Ala is present at C-terminus and the presence of APyC at the C-terminus inhibits the formation of rearrangement ions. In addition, abundant b_n–1⁺ ions are formed, presumably with stable oxazolone structures, when the C-terminus of b_n–1⁺ ions possessed D-Ala.

CONCLUSIONS

The present study demonstrates that ESI tandem mass spectrometry is very useful for differentiating positional isomers of hybrid peptides containing D-Ala and APyC amino acids. While the protonated peptides give rise to characteristic sequencing ions, the cationized peptides produce additional rearrangement ions ([b_n–1 + OCH₃ + Na]⁺ and [b_n–1 + OH + Na]⁺) which helps distinguish between the presence of D-Ala and APyC amino acids at the C-terminus. Copyright © 2012 John Wiley & Sons, Ltd.