Side Chain Driven Mass Spectral Fragmentation of Lys Containing Peptides: Cα‑CO Bond Cleavage in Xxx-Lys-Xxx Sequences

Mass spectral fragmentation of the tripeptide amide Ala-Lys-Ala-amide (AKA*) yields a product ion at m/z 228.1, which corresponds to a neutral loss of 43 Da from the b3 ion (m/z 271.1). Similar losses of 43 Da are observed from bn ions in the hexapeptide AKAAKA* and tetrapeptide AKAA*. The role of the Lys2 residue, in mediating the neutral loss, is supported by the absence of the corresponding product ion in the MS2 spectrum of AVA* and attenuation of the intensity in analogs containing ornithine/diaminobutyric acid residues at position 2. The role of the N-terminus amino group is suggested by the absence of this neutral loss when the residue Ala1 amino group is acetylated or dimethylated. In XKA* sequences, where X = Gly, Ala, Leu, Aib and Pro, the observed neutral losses from b3 are 29, 43, 85, 57, and 69 Da, respectively, indicative of Cα-CO bond cleavage releasing the R-CH = NH fragment. Isotope labeling and comparison with mass spectral fragments generated from synthetic Nα-formyl Lys-Ala-amide (fKA*) establish the identity of the ion at m/z 228 in the MS2 spectrum of AKA* as the b ion [fKA]. A charge remote fragmentation pathway, involving proton abstraction from the terminal amino group, by the Lys2 ε amino group, followed by Cα-CO bond cleavage, is proposed as a plausible mechanism for the observed noncanonical cleavage process.