New strategies for profiling and characterization of human milk oligosaccharides.

Human breast milk is an incredibly rich and complex biofluid composed of proteins, lipids and complex carbohydrates, including a diverse repertoire of free human milk oligosaccharides (HMOs). Strikingly, HMOs are not digested by the infant but function as prebiotics for bacterial strains associated with numerous benefits. Considering the broad variety of beneficial effects of HMOs, and the vast number of factors that affect breast milk composition, the analysis of HMO diversity and complexity is of utmost relevance. Using human milk samples from a cohort of Bangladeshi mothers participating in a study on malnutrition and stunting in children, we have characterized breast milk oligosaccharide composition by means of permethylation followed by liquid chromatography coupled with high-resolution tandem mass spectrometry (LC-MS/MS) analysis. This approach identified over 100 different glycoforms and showed a wide diversity of milk composition, with a predominance of fucosylated and sialylated HMOs over nonmodified HMOs. We observed that these samples contain on average 80 HMOs, with the highest permethylated masses detected being >5000 mass units. Here we report an easily implemented method developed for the separation, characterization and relative quantitation of large arrays of HMOs, including higher molecular weight sialylated HMOs. Our ultimate goal is to create a simple, high-throughput method, which can be used for full characterization of sialylated and/or fucosylated HMOs. These results demonstrate how current analytical techniques can be applied to characterize human milk composition, providing new tools to help the scientific community shed new light on the impact of HMOs during infant development.