Electrospray Ionization Tandem Mass Spectrometric Study of Protonated and Alkali-Cationized α/ε-Hybrid Peptides: Differentiation of a Pair of Dipeptide Positional Isomers

A new class of Boc-N-protected hybrid peptides derived from L-Ala and ε6-Caa (L-Ala = L-Alanine, Caa = C-linked carboamino acid derived from D-xylose) have been studied by positive ion electrospray ionization (ESI) ion-trap tandem mass spectrometry (MS/MS). MS n spectra of protonated and alkali-cationized hybrid peptides produce characteristic fragmentation involving the peptide backbone, the tert-butyloxycarbonyl (Boc) group, and the side chain. The dipeptide positional isomers are differentiated by the collision-induced dissociation (CID) of the protonated and alkali-cationized peptides. The CID of [M + H]+ ion of Boc-NH-L-Ala-ε-Caa-OCH3 (1) shows a prominent [M + H – C4H8]+ ion, which is totally absent for its positional isomer Boc-NH-ε-Caa-L-Ala-OCH3 (6), which instead shows significant loss of t-butanol. The formation of the [M + Cat – C4H8]+ ion is totally absent and [M + Cat – Boc + H]+ is prominent in the CID of the [M + Cat]+ ion of Boc-NH-L-Ala-ε-Caa-OCH3 (1), whereas the former is highly abundant and the latter is of low abundance for its positional isomer Boc-NH-ε-Caa-L-Ala-OCH3 (6). It is observed that ‘b’ ions are abundant when oxazolone structures are formed through a five-membered cyclic transition state in tetra-, penta-, and hexapeptides and the cyclization process for larger ‘b’ ions led to an insignificant abundance. However, the significant ‘b’ ion is formed in ε,α-dipeptide, which may have a seven-membered substituted 2-oxoazepanium ion structure. The MS n spectra of [M + Cat – Boc + H]+ ions of these peptides are found to be significantly different to those of [M + H – Boc + H]+ ions. The CID spectra of [M + Cat – Boc + H]+ ions of peptide acids containing L-Ala at the C-terminus show an abundant N-terminal rearrangement ion, [b n + 17 + Cat]+, which is absent for the peptide acids containing ε-Caa at the C-terminus. Thus, the results of these hybrid peptides provide sequencing information, the structure of the cyclic intermediate involved in the formation of the rearrangement ion, and distinguish a pair of dipeptide positional isomers.