title: Comparative analysis of native and permethylated human milk oligosaccharides by liquid chromatography coupled to high resolution mass spectrometry.
abstract: Human milk oligosaccharides (HMOs) represent the third most abundant components of milk after lactose and lipids. HMOs are indigestible by the suckling infant but can act as prebiotics and have significant biological functions regarding the organism defense against pathogens (such as bacteria or viruses) by preventing interactions with their receptors. Although constituted of only five distinct monosaccharide building blocks, HMOs are highly structurally diverse compounds with many co-existing structural isomers. Here we report the development and comparison of two distinct glycomic platforms based on liquid chromatography coupled to high resolution mass spectrometry (LC-MS) for analyzing HMOs. We have implemented and thoroughly compared the LC-MS of permethylated and native HMOs on reversed phase (RP) and porous graphitic carbon (PGC) columns for their ability to resolve the natural heterogeneity of milk oligosaccharides at the highest sensitivity. Our data essentially underlines the usefulness of analyzing HMOs as permethylated derivatives especially for getting more precise structural information at high sensitivity. For instance, permethylation annihilates gas-phase fucose migration during MS/MS experiments, thus facilitating spectra interpretation and giving access to relevant information regarding oligosaccharide branching and isomer distinction. At the opposite, LC-MS profiling of native HMOs (using PGC) in milk performed best in terms of detected species, while also being much faster in terms of sample preparation. Although less efficient than PGC chromatography, RPLC proved successful for separating pairs of permethylated isomeric HMOs. A key advantage of RP over PGC liquid chromatography is that retention times can be correlated to molecular weights, which can greatly facilitate further HMO identification using retention time prediction. Altogether these data lead us to think that LC-MS analysis of native HMOs (using PGC) can be used as first-line profiling approach while permethylation can be performed afterwards for facilitating structural characterization.
authors.affiliation: CEA, IBITECS, Service de Pharmacologie et d'Immunoanalyse, UMR 0496, Laboratoire d'Etude du Métabolisme des Médicaments, MetaboHUB-Paris, Université Paris Saclay, F-91191 Gif-sur-Yvette cedex, France.
authors.forename: Stéphanie
authors.initials: S
authors.lastname: Oursel
authors.affiliation: CEA, IBITECS, Service de Pharmacologie et d'Immunoanalyse, UMR 0496, Laboratoire d'Etude du Métabolisme des Médicaments, MetaboHUB-Paris, Université Paris Saclay, F-91191 Gif-sur-Yvette cedex, France.
authors.forename: Sophie
authors.initials: S
authors.lastname: Cholet
authors.affiliation: CEA, IBITECS, Service de Pharmacologie et d'Immunoanalyse, UMR 0496, Laboratoire d'Etude du Métabolisme des Médicaments, MetaboHUB-Paris, Université Paris Saclay, F-91191 Gif-sur-Yvette cedex, France.
authors.forename: Christophe
authors.initials: C
authors.lastname: Junot
authors.affiliation: CEA, IBITECS, Service de Pharmacologie et d'Immunoanalyse, UMR 0496, Laboratoire d'Etude du Métabolisme des Médicaments, MetaboHUB-Paris, Université Paris Saclay, F-91191 Gif-sur-Yvette cedex, France. Electronic address: francois.fenaille@cea.fr.
authors.forename: François
authors.initials: F
authors.lastname: Fenaille
mesh_terms: D002853:Chromatography, Liquid / Q000379:methods*; D006801:Humans; D007536:Isomerism; D013058:Mass Spectrometry / Q000379:methods*; D008895:Milk, Human / Q000737:chemistry*; D009844:Oligosaccharides / Q000032:analysis / Q000737:chemistry / Q000302:isolation & purification
chemical_list: D009844:Oligosaccharides
keywords: High resolution mass spectrometry; Human milk oligosaccharides; Permethylation; Porous graphitic carbon