Identification and accurate quantitation of biological oligosaccharide mixtures

ABSTRACT: Structure-specific characterization and quantitation is often required for effective functional studies of oligosaccharides. Inside the gut, HMOs are preferentially bound and catabolized by the beneficial bacteria. HMO utility by these bacteria employs structure-specific catabolism based on a number of glycosidases. Determining the activity of these enzymes requires accurate quantitation of a large number of structures. In this study, we describe a method for the quantitation of human milk oligosaccharide (HMO) structures employing LC/MS and isotopically labeled internal standards. Data analysis was accomplished with a newly developed software tool, LC/MS Searcher, that employs a reference structure library to process LC/MS data yielding structural identification with accurate quantitation. The method was used to obtain a meta-enzyme analysis of bacteria, the simultaneous characterization of all glycosidases employed by bacteria for the catabolism of milk oligosaccharides. Analysis of consumed HMO structures confirmed the utility of a β-1,3-galactosidase in Bif idobacterium longum subsp. infantis ATCC 15697 (B. infantis). In comparison, Bif idobacterium breve ATCC 15700 showed significantly less HMO catabolic activity compared to B. infantis. Human milk oligosaccharides (HMOs) are an importantcomponent of breast milk and provide health benefits that infant formula manufacturers want to mimic; however, th