CE-MS Identification of Amino Acid Sequence Inversion as a New Degradation Pathway of an Aspartyl Model Tripeptide

CE-MS was employed to identify two unknown degradation products of the model tripeptide Phe-α-Asp-Gly heated at 80 °C in aqueous solution at pH 7.4. Both compounds displayed essentially identical mass spectra indicating the presence of peptide diastereomers. The M + H]⁺-ion at m/z 338 suggested a tripeptide composed of the amino acids Phe, Gly and Asp. The fragmentation pattern indicated that Phe was not located at the N-terminus. Subsequently, the linear peptide α-Asp-Phe-Gly and the branched peptide Asp(Gly)-Phe were synthesized and analyzed by CE-MS. The mass spectrum of synthetic α-Asp-Phe-Gly was identical to that of the unknown compounds confirming the structure of the degradation products. Asp(Gly)-Phe displayed a complex fragmentation pattern. In conclusion, amino acid sequence inversion represents another degradation pathway of Phe-α-Asp-Gly at pH 7.4 besides known reactions including isomerization, enantiomerization, cyclization to diketopiperazine derivatives and backbone hydrolysis. The mechanism of the rearrangement of the amino acid sequence is proposed to proceed via an aza-bridged intermediate.