title: New features of site-specific horseradish peroxidase (HRP) glycosylation uncovered by nano-LC-MS with repeated ion-isolation/fragmentation cycles.
abstract: Horseradish peroxidase (HRP) is widely used in biomedical research as a reporter enzyme in diagnostic assays. In addition, it is of considerable interest as a model glycoprotein with core-xylosylated and -(alpha1-3)-fucosylated N-glycans that form antigenic elements of plant allergens and parasitic helminths. Using a combination of techniques comprising (1) nano-liquid chromatography (LC)-mass spectrometry (MS)/MS with multiple selection/fragmentation cycles of HRP tryptic (glyco-)peptides, (2) nano-electrospray MS of intact HRP, and (3) carbohydrate linkage analysis, it was revealed that most of the HRP N-glycosylation sites can be occupied with an alternative Fuc(1-3)GlcNAc-disaccharide. Two main variants of HRP occur: The major population (approximately 60%) has eight glycosylation sites carrying core(1-3)fucosylated, xylosylated, trimannosyl N-glycans, with the ninth potential N-glycosylation site Asn316 not occupied. Another group of HRP carries seven of the above-mentioned N-glycans, with an eighth N-glycosylation site carrying the alternative Fuc(1-3)GlcNAc-unit (approximately 35%). In addition, minor subsets of HRP were found to contain a xylosylated, trimannosyl N-glycan lacking core-fucosylation as a ninth N-glycan attached to Asn316, which has hitherto been assumed to be unoccupied. The finding of these new features of glycosylation of an already exceptionally well-studied glycoprotein underscores the potential of the nano-LC-MS(n) based analytical approach followed.
authors.affiliation: Biomolecular Mass Spectrometry Unit, Department of Parasitology, Center of Infectious Diseases, Leiden University Medical Center, P.O. Box 9600, 2300 RC Leiden, The Netherlands. m.wuhrer@lumc.nl
authors.forename: Manfred
authors.initials: M
authors.lastname: Wuhrer
authors.forename: Crina I A
authors.initials: CI
authors.lastname: Balog
authors.forename: Carolien A M
authors.initials: CA
authors.lastname: Koeleman
authors.forename: André M
authors.initials: AM
authors.lastname: Deelder
authors.forename: Cornelis H
authors.initials: CH
authors.lastname: Hokke
mesh_terms: D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid; D008401:Gas Chromatography-Mass Spectrometry; D006031:Glycosylation; D006735:Horseradish Peroxidase / Q000737:chemistry / Q000378:metabolism*; D013058:Mass Spectrometry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D036103:Nanotechnology
chemical_list: D006735:Horseradish Peroxidase