title: Human Milk Oligosaccharide Specificities of Human Galectins. Comparison of Electrospray Ionization Mass Spectrometry and Glycan Microarray Screening Results.
abstract: The affinities of thirty-two free human milk oligosaccharides (HMOs) for four human galectin proteins, a stable mutant of hGal1 (hGal-1), a C-terminal fragment of hGal-3 (hGal-3C), hGal-7, and an N-terminal fragment of hGal-9 (hGal-9N), were measured using electrospray ionization mass spectrometry (ESI-MS). The binding data show that each of the four galectins recognize the majority of the HMOs tested (hGal-1 binds thirty-two HMOs, hGal-3C binds twenty-six, hGal-7 binds thirty-one, and hGal-9N binds twenty-six). Twenty-five of the HMOs tested bind all four galectins, with affinities ranging from 103to 105M-1. The reliability of the ESI-MS assay for quantifying the affinities of HMOs for lectins was established from the agreement found between the ESI-MS data and affinities of a small number of HMOs for hGal-1, hGal-3C, and hGal-7 measured by isothermal titration calorimetry (ITC). Comparison of the relative affinities (of 14 HMOs) measured by ESI-MS with the reported specificities of hGal-1, hGal-3, hGal-7, and hGal-9 for these same HMOs established using the shotgun human milk glycan microarray (HM-SGM-v2) showed fair-to-poor correlation, with evidence of false positives and false negatives in the microarray data. The results of this study suggest that HMO specificities of lectins established using microarrays may not accurately reflect their true HMO-binding properties and that the use of "in solution" assays such as ESI-MS and ITC is to be preferred.
authors.affiliation: Alberta Glycomics Centre and Department of Chemistry, University of Alberta , Edmonton, Alberta Canada T6G 2G2.
authors.forename: Km
authors.initials: K
authors.lastname: Shams-Ud-Doha
authors.affiliation: Alberta Glycomics Centre and Department of Chemistry, University of Alberta , Edmonton, Alberta Canada T6G 2G2.
authors.forename: Elena N
authors.initials: EN
authors.lastname: Kitova
authors.affiliation: Alberta Glycomics Centre and Department of Chemistry, University of Alberta , Edmonton, Alberta Canada T6G 2G2.
authors.forename: Pavel I
authors.initials: PI
authors.lastname: Kitov
authors.affiliation: INRS-Institut Armand-Frappier , Laval, Québec Canada H7 V 1B7.
authors.forename: Yves
authors.initials: Y
authors.lastname: St-Pierre
authors.affiliation: Alberta Glycomics Centre and Department of Chemistry, University of Alberta , Edmonton, Alberta Canada T6G 2G2.
authors.forename: John S
authors.identifier: 0000-0002-3389-7112
authors.initials: JS
authors.lastname: Klassen
mesh_terms: D002151:Calorimetry; D037161:Galectins / Q000378:metabolism*; D006801:Humans; D046228:Microarray Analysis; D008895:Milk, Human / Q000737:chemistry*; D009844:Oligosaccharides / Q000378:metabolism*; D010446:Peptide Fragments / Q000378:metabolism; D011485:Protein Binding; D021241:Spectrometry, Mass, Electrospray Ionization
chemical_list: D037161:Galectins; D009844:Oligosaccharides; D010446:Peptide Fragments