Can α- and β-Alanine Containing Peptides Be Distinguished Based on the CID Spectra of Their Protonated Ions?

The fragmentation reactions of isomeric dipeptides containing α- and β-alanine residues (αAla-αAla, αAla-βAla, βAla-αAla, and βAla-βAla) were studied using a combination of low-energy and energy resolved collision induced dissociation (CID). Each dipeptide gave a series of different fragment ions, allowing for differentiation. For example, peptides containing an N-terminal β-Ala residue yield a diagnostic imine loss, while lactam ions at m/z 72 are unique to peptides containing β-Ala residues. In addition, MS³ experiments were performed. Structure-specific fragmentation reactions were observed for y₁ ions, which help identify the C-terminal residue. The MS³ spectra of the b₂ ions are different suggesting they are unique for each peptide. Density functional theory (DFT) calculations predict that b₂ ions formed via a neighboring group attack by the amide are thermodynamically favored over those formed via neighboring group attack by the N-terminal amine. Finally, to gain further insight into the unique fragmentation chemistry of the peptides containing an N-terminal β-alanine residue, the fragmentation reactions of protonated β-Ala-NHMe were examined using a combination of experiment and DFT calculations. The relative transition-state energies involved in the four competing losses (NH₃, H₂O, CH₃NH₂, and CH₂=NH) closely follow the relative abundances of these as determined via CID experiments.