Recent advances on separation and characterization of human milk oligosaccharides

Abstract

Free human milk oligosaccharides (HMOs) are unique due to their highly complex nature and important emerging biological and protective functions during early life such as prebiotic activity, pathogen deflection, and epithelial and immune cell modulation. Moreover, four genetically determined heterogeneous HMO secretory groups are known to be based on their structure and composition. Over the years, several analytical techniques have been applied to characterize and quantitate HMOs, including nuclear magnetic resonance spectroscopy, high-performance liquid chromatography (HPLC), high pH anion-exchange chromatography, off-line and on-line mass spectrometry (MS), and capillary electrophoresis (CE). Even if these techniques have proven to be efficient and simple, most glycans have no significant UV absorption and derivatization with fluorophore groups prior to separation usually results in higher sensitivity and an improved chromatographic/electrophoretic profile. Consequently, the analysis by HPLC/CE of derivatized milk oligosaccharides with different chromophoric active tags has been developed. However, UV or fluorescence detection does not provide specific structural information and this is a key point in particular related to the highly complex nature of the milk glycan mixtures. As a consequence, for a specific determination of complex mixtures of oligomers, analytical separation is usually required with evaluation by means of MS, which has been successfully applied to HMOs, resulting in efficient compositional analysis and profiling in various milk samples. This review aims to give an overview of the current state-of-the-art techniques used in HMO analysis.

Separation of isomeric glycans by ion mobility spectrometry – the impact of fluorescent labelling

“…These structures are typically found as features on larger glycoconjugates such a N-and O-linked glycans or on human milk oligosaccharides (HMO). 24 The two main types of Lewis antigens are the tetrasaccharide motifs LeY and LeB, which feature a common monosaccharide composition and are displayed in Fig. 1.…”

Unraveling the isomeric heterogeneity of glycans: ion mobility separations in structures for lossless ion manipulations

“…HMOs have received widespread attention for their roles in potentially protecting the gut microbiome, promoting growth and immunological development in infants, and their anti-adhesive and probiotic properties 6 , 11 . Lacto-N-tetraose and lacto-N-neotetraose (LNT and LNnT, respectively), which only differ in their linkage position (1–3 versus 1–4) between the D-galactose and N-acetyl D-glucosamine residues, were able to be baseline resolved after 31.5 meters of separation as their protonated adducts (Figure 4).…”

Elucidating Human Milk Oligosaccharide biosynthetic genes through network-based multiomics integration

“…Enumerating all feasible permutations of the elementary reaction ( Figure 3A; S1.1.1), we delineated every possible reaction series from lactose to each of the 16 most abundant HMOs. Of the measured HMOs, 11 have fully determined molecular structures, while the remaining five have multiple candidate structures ( Figure 1C, Figure S 12) 6,8,34,[99][100][101] . The set of all possible reactions leading to characterized and ambiguous structures formed the Complete Network ( Figure 3B; Supplemental Methods S1.1.1).…”