Barcoding for Isobaric Tagging of Carboxylic Acids and Amines Metabolites

Abstract

Conventional isobaric platforms in metabolomic analyses are designed to label a single functional group, typically primary amines. This specificity restricts the range of detectable analytes in a single injection, limiting overall chemical coverage of metabolites, and potentially reducing the depth of biological insight. Using a proof-of-concept 2-plex isobaric barcode tagging scheme, amine-containing metabolites are selectively labeled using acid barcode tags. Acid-containing metabolites (carboxylate) are derivatized using amine barcode tags. This allows for broad coverage of two metabolite classes in a single injection in a 30-minute LC gradient. These barcode tags undergo double fragmentation to generate cyclized products and distinct reporter ions that are specific to each metabolite class, enabling accurate quantitation. This tagging scheme also serves as an untargeted approach for identifying metabolites not present in the user’s metabolic library. Tagged acid and tagged amine analytes are mixed at ratios of 1:2:5:10 using various amounts of Escherichia coli lysate to produce a 10-fold linear dynamic range, an average linearity (R²) of 0.99, and an average RSD of 2.64%. Metabolic changes in amine and carboxylic acid metabolism of genetic knockout PanC and wild-type Escherichia coli were characterized using this method.