title: Use of Wisteria floribunda agglutinin affinity chromatography in the structural analysis of the bovine lactoferrin N-linked glycosylation.
abstract: BACKGROUND
  Over the years, the N-glycosylation of both human and bovine lactoferrin (LF) has been studied extensively, however not all aspects have been studied in as much detail. Typically, the bovine LF complex-type N-glycans include certain epitopes, not found in human LF N-glycans, i.e. Gal(α1-3)Gal(β1-4)GlcNAc (αGal), GalNAc(β1-4)GlcNAc (LacdiNAc), and N-glycolylneuraminic acid (Neu5Gc). The combined presence of complex-type N-glycans, with αGal, LacdiNAc, LacNAc [Gal(β1-4)GlcNAc], Neu5Ac (N-acetylneuraminic acid), and Neu5Gc epitopes, and oligomannose-type N-glycans complicates the high-throughput analysis of such N-glycoprofiles highly.
  
  
  METHODS
  For the structural analysis of enzymatically released N-glycan pools, containing both LacNAc and LacdiNAc epitopes, a prefractionation protocol based on Wisteria floribunda agglutinin affinity chromatography was developed. The sub pools were analysed by MALDI-TOF-MS and HPLC-FD profiling, including sequential exoglycosidase treatments.
  
  
  RESULTS
  This protocol separates the N-glycan pool into three sub pools, with (1) free of LacdiNAc epitopes, (2) containing LacdiNAc epitopes, partially shielded by sialic acid, and (3) containing LacdiNAc epitopes, without shielding by sialic acid. Structural analysis by MALDI-TOF-MS and HPLC-FD showed a complex pattern of oligomannose-, hybrid-, and complex-type di-antennary structures, both with, and without LacdiNAc, αGal and sialic acid.
  
  
  CONCLUSIONS
  Applying the approach to bovine LF has led to a more detailed N-glycome pattern, including LacdiNAc, αGal, and Neu5Gc epitopes, than was shown in previous studies.
  
  
  CONCLUSIONS
  Bovine milk proteins contain glycosylation patterns that are absent in human milk proteins; particularly, the LacdiNAc epitope is abundant. Analysis of bovine milk serum proteins is therefore excessively complicated. The presented sub fractionation protocol allows a thorough analysis of the full scope of bovine milk protein glycosylation. This article is part of a Special Issue entitled Glycoproteomics.
authors.affiliation: Department of Microbial Physiology, Groningen Biomolecular Sciences and Biotechnology Institute (GBB), University of Groningen, Nijenborgh 7, NL-9747 AG Groningen, The Netherlands.
authors.forename: Sander S
authors.initials: SS
authors.lastname: van Leeuwen
authors.forename: Ruud J W
authors.initials: RJ
authors.lastname: Schoemaker
authors.forename: Christel J A M
authors.initials: CJ
authors.lastname: Timmer
authors.forename: Johannis P
authors.initials: JP
authors.lastname: Kamerling
authors.forename: Lubbert
authors.initials: L
authors.lastname: Dijkhuizen
mesh_terms: D000818:Animals; D002240:Carbohydrate Sequence; D002417:Cattle; D002846:Chromatography, Affinity / Q000379:methods*; D006031:Glycosylation; D007781:Lactoferrin / Q000032:analysis / Q000737:chemistry* / Q000378:metabolism*; D008892:Milk / Q000737:chemistry / Q000378:metabolism; D008954:Models, Biological; D008969:Molecular Sequence Data; D015394:Molecular Structure; D037121:Plant Lectins / Q000494:pharmacology*; D011134:Polysaccharides / Q000032:analysis / Q000737:chemistry / Q000378:metabolism*; D034781:Receptors, N-Acetylglucosamine
chemical_list: D037121:Plant Lectins; D011134:Polysaccharides; D034781:Receptors, N-Acetylglucosamine; C040129:wisteria lectin; D007781:Lactoferrin