New strategies for profiling and characterization of human milk oligosaccharides

Abstract

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.

Keywords: CID; LC-NSI-MS/MS; MALDI-TOF-MS; human milk oligosaccharides (HMOs); structural analysis.

Fig. 1

Structural diversity of human milk oligosaccharides. With very few exceptions, all HMOs are formed by a lactose core (Gal-β1 → 4-Glc, center), which can be extended enzymatically in repeats of lacto-N-biose (Gal-β1 → 3-GlcNAc) or N-acetyllactosamine (Gal-β1 → 4-GlcNAc) (upper right). Lactose can also be fucosylated or sialylated in different linkages (upper left). Linkage and position of fucose residues generate isomers, which can result in structures that are phenotypically related to secretor status and Lewis blood group (lower left). Isomerization can also occur as a result of sialylation of elongated core structures (lower center). HMOs can also be simultaneously fucosylated and sialylated, resulting in highly complex structures (lower right). The monosaccharide key is shown at the bottom. Abbreviations: 2′-FL, 2′-fucosyllactose; 3-FL, 3-fucosyllactose; 3′-SL, 3′-sialyllactose; 6′-SL, 6′-sialyllactose; LNFP I, II, III, V, lacto-N-fucopentaose I, II, III, V; LNT, lacto-N-tetraose; LNnT, lacto-N-neotetraose; LNH, lacto-N-hexaose; LNnH, lacto-N-neohexaose; pLNH, para-lacto-N-hexaose; LST a, b, c, sialyl-lacto-N-tetraoses a-c; S-LNF II or F-LST a, sialylfucosyllacto-N-tetraose; DS-LNF II or FDS-LNT I, fucosyldisialyllacto-N-tetraose; TetraF-LND III, tetrafucosyl-lacto-N-decaose III. Monosaccharide symbols follow the Symbol Nomenclature for Graphical Representation of Glycans (https://www.ncbi.nlm.nih.gov/glycans/snfg.html). This figure is available in black and white in print and in colour at Glycobiology online.

Fig. 2

Representative MALDI-TOF-MS spectrum of permethylated human milk oligosaccharides. All masses correspond to fully permethylated, free reducing end, sodium adducts of HMOs. Possible structures for each mass are shown. Free lactose was excluded from the spectrum. HMOs were identified using Glycomod (web.expasy.org/glycomod) as a search engine. This figure is available in black and white in print and in colour at Glycobiology online.

Fig. 3

LC-MS separation of permethylated human milk oligosaccharides. (A) Full MS scans at 17 min and 26 min of elution time. For the first 5 min of the separation, the main eluting compound is free lactose (top). After 20 min (bottom), HMOs start to elute and can be isolated from lactose. Several multiply charged ions can be seen. (B) EICs corresponding to the most abundant charge state of some of the most abundant HMOs found in the samples. The TIC trace (shown on top) shows the full elution profile, where the main peak corresponds to the elution of lactose (full MS shown in A). Mass accuracy: 5 ppm. This figure is available in black and white in print and in colour at Glycobiology online.

Fig. 4

Examples of diagnostic fragment ions generated by permethylated HMOs. Permethylation provides structural information because it allows distinguishing internal, terminal and reducing end fragments. This figure is available in black and white in print and in colour at Glycobiology online.

Fig. 5

Structure assignment based on MS/MS analysis. CID MS² spectra of (A) m/z 651.32, (B) m/z 838.40, (C) m/z 1100.55 and (D) m/z 1549.77. Permethylated fragments aid in structure determination and evaluation of number of isomers for a given mass. Fragments marked with asterisk represent fucose linkage isomers. This figure is available in black and white in print and in colour at Glycobiology online.

Fig. 6

Isomer distinction based on chromatographic separation and MS/MS fragmentation. CID MS² spectra of EIC peaks of [M + Na]⁺ 2085.04, eluting at 28.45 and 35.65 min. Characteristic fragments that allow for isomer(s) identification are highlighted. This figure is available in black and white in print and in colour at Glycobiology online.