Unusual fragmentation of β-linked peptides by ExD tandem mass spectrometry

Ion-electron reaction based fragmentation methods (ExD) in tandem mass spectrometry (MS), such as electron capture dissociation (ECD) and electron transfer dissociation (ETD) represent a powerful tool for biological analysis. ExD methods have been used to differentiate the presence of the isoaspartate (isoAsp) from the aspartate (Asp) in peptides and proteins. IsoAsp is a β(3)-type amino acid that has an additional methylene group in the backbone, forming a C(α)-C(β) bond within the polypeptide chain. Cleavage of this bond provides specific fragments that allow differentiation of the isomers. The presence of a C(α)-C(β) bond within the backbone is unique to β-amino acids, suggesting a similar application of ExD toward the analysis of peptides containing other β-type amino acids. In the current study, ECD and ETD analysis of several β-amino acid containing peptides was performed. It was found that N-C(β) and C(α)-C(β) bond cleavages were rare, providing few c and z• type fragments, which was attributed to the instability of the C(β) radical. Instead, the electron capture resulted primarily in the formation of a• and y fragments, representing an alternative fragmentation pathway, likely initiated by the electron capture at a backbone amide nitrogen protonation site within the β amino acid residues.