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

Abstract

AbstractAcetylation 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 Nα‐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 Nα‐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 Nα‐acetyl and side‐chain acetyl amino acids.