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 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 Bifidobacterium longum subsp. infantis ATCC 15697 (B. infantis). In comparison, Bifidobacterium breve ATCC 15700 showed significantly less HMO catabolic activity compared to B. Infantis.

Variation in Consumption of Human Milk Oligosaccharides by Infant Gut-Associated Strains of Bifidobacterium breve

“…Figure 3B presents the consumption of neutral nonfucosylated HMO in more detail, showing that consumption patterns among the B. breve strains were similar. All strains were able to deplete LNT/LNnT from the culture medium to a high extent as previously witnessed (17). Among three major hexaoses found in HMO, a preference for lacto-N-neohexaose (LNnH) was observed over lacto-N-hexaose (LNH) and para-lacto-N-hexaose (p-LNH).…”

“…infantis ATCC 15697 (64% consumption) but clearly higher than for B. breve ATCC 15700 ( Fig. 2A), the only B. breve strain glycoprofiled to date (17,29,30). Figure 3B presents the consumption of neutral nonfucosylated HMO in more detail, showing that consumption patterns among the B. breve strains were similar.…”

“…Only one strain of B. breve, ATCC 15700, has been shown to utilize a far less diverse range of HMO isomers than B. longum subsp. infantis ATCC 15697 (17). Whether this pattern is representative of the B. breve species is not clear.…”

Challenges in Separations of Proteins and Small Biomolecules and the Role of Modern Mass Spectroscopy Tools for Solving Them, as Well as Bypassing Them, in Structural Analytical Studies of Complex Biomolecular Mixtures

“…Note however that this separation technique has still not been applied/modified for purification of HMOs, which is very important for the MS research field, both basic and applied. Over the last decade, Lebrilla et al developed an LC-MS method for the analysis of HMO samples, based on a nanoHPLC Chip/TOF MS approach [7,8,14,15,22,[50][51][52] using graphitized carbon chromatography (GCC). GCC is stable, has a long life [53] and is effective for the separation of both neutral and acidic oligosaccharides, including isomers [54].…”

“…This has been long true for peptides [80], while the respective methods have been extensively refined over the last decade for glycans [14,15,22,52], and lipids [81]. Aside from efficiently dealing with separation of isomeric structures, LC is not hindered with small amounts of salts, detergents or buffers.…”