Characterization of isomeric acetyl amino acids and di-acetyl amino acids by LC/MS/MS

Abstract

Acetylation of amino acids is important in the molecular biology and biochemistry because they are part of several metabolic pathways. N-acetyl amino acids can form through degradation of N-acetyl proteins or direct acetylation of amino acids by specific enzymes. Acetylation of α-amino acids can be either on the alpha –NH₂ or on the side-chain functional group, where both the acetyl products are isomeric and can show different biological roles. Theoretically, all proteinogenic α-amino acids are expected to undergo acetylation and they can be a part of metabolome. Thus, it is essential to detect and identify all the possible acetylated products of α-amino acids for untargeted metabolomics studies. In this study, it is aimed to synthesize and characterize all acetylated products of natural α-amino acids. A total of 20 N_α-acetyl amino acids (1–20), six side-chain acetyl amino acids (21–26), and six diacetyl amino acids (27–32) were synthesized and characterized by liquid chromatography-electrospray ionizationtandem mass spectrometry (LC–ESI–MS/MS). The [M + H]⁺ ions of all the acetyl amino acids were subjected to MS/MS experiments to obtain their structural information. Apart from the expected loss of (H₂O + CO) (immonium ions), most of the acetyl amino acids specifically showed loss of H₂O and loss of a ketene (C₂H₂O) from [M+H]⁺ ions. The side-chain acetyl amino acids showed a clear-cut structure specific fragment ions that enabled easy differentiation from their isomeric N_α-acetyl amino acids. The other isomeric/isobaric acetyl amino acids could also be easily distinguished by their MS/MS spectra. The MS/MS of immonium ions of the acetyl amino acids were also studied, and they included characteristic products reflecting the structures of parent N_α-acetyl and side-chain acetyl amino acids.