Glycans constitute a new class of compounds for biomarker discovery. Glycosylation is a common post-translational modification and is often associated with transformation to malignancy. To analyze glycans, they are released from proteins, enriched, and measured with mass spectrometry. For biomarker discovery, repeatability at every step of the process is important. Locating and minimizing the process variability is key to establishing a robust platform stable enough for biomarker discovery. Understanding the variability of the measurement devices helps understand the variability associated with the chemical processing. This report explores the potential use of methods expediting the enzymatic release of glycans such as a microwave reactor and automation of the solid-phase extraction with a robotic liquid handler. The study employs matrix-assisted laser desorption/ ionization -Fourier transform ion cyclotron resonance mass spectrometry but would be suitable with any mass spectrometry method. Methods for system-wide data analysis are examined because proper metrics for evaluating the performance of glycan sample preparation procedures are not well established.

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.

Prebiotics are non-digestible substrates that stimulate the growth of beneficial microbes in the human intestine. Galactooligosaccharides (GOS) are food ingredients that possess prebiotic properties, in particular, promoting the growth of bifidobacteria in situ. However precise mechanistic details of GOS consumption by bifidobacteria remains poorly understood. Because GOS are mixtures of polymers of different lengths and linkages, there is interest to determine which specific structures provide prebiotic effects in order to potentially create better supplements. We here present a method comprising porous graphitic carbon separation, isotopic labeling and mass spectrometry analysis for the structure specific analysis of GOS isomers and their bacterial consumption rate. Using this strategy, the differential bacterial consumption of GOS by the bifidobacteria species B. longum subsp. infantis, B. animalis subsp. lactis and B. adolescentis was determined, indicating that the use of specific GOS isomers in infant formula may provide enrichment of distinct species.

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