title: Label-free glycoprofiling with multiplex surface plasmon resonance: a tool to quantify sialylation of erythropoietin.
abstract: Protein glycosylation is among the most common and well-defined post-translational modifications due to its vital role in protein function. Monitoring variation in glycosylation is necessary for producing more effective therapeutic proteins. Glycans attached to glycoproteins interact highly specific with lectins, natural carbohydrate-binding proteins, which property is used in the current label-free methodology. We have established a lectin microarray for label-free detection of lectin-carbohydrate interactions allowing us to study protein glycosylation directly on unmodified glycoproteins. The method enables simultaneous measurement of up to 96 lectin-carbohydrate interactions on a multiplex surface plasmon resonance imaging platform within 20 min. Specificity determination of lectins succeeded by analysis of neoglycoproteins and enzymatically remodeled glycoproteins to verify carbohydrate binding. We demonstrated the possibilities for glycosylation fingerprinting by comparing different Erythropoietin sources without the need for any sample pretreatment and we were able to accurately quantify relative sialylation levels of Erythropoietin.
authors.affiliation: †Downstream Processing, Synthon Biopharmaceuticals BV, P.O. Box 7071, 6503 GN Nijmegen, The Netherlands.|‡Bioprocess Engineering, Wageningen University, P.O. Box 16, 6700 AA Wageningen, The Netherlands.
authors.forename: Karin P M
authors.initials: KP
authors.lastname: Geuijen
authors.affiliation: §Department of Pharmaceutics, Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Universiteitsweg 99, 3584 CG Utrecht, The Netherlands.
authors.forename: Liem A
authors.initials: LA
authors.lastname: Halim
authors.affiliation: §Department of Pharmaceutics, Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Universiteitsweg 99, 3584 CG Utrecht, The Netherlands.
authors.forename: Huub
authors.initials: H
authors.lastname: Schellekens
authors.affiliation: ∥IBIS Technologies, Pantheon 5, 7521 PR Enschede, The Netherlands.|⊥Medical Cell Biophysics Group, MIRA Institute, Faculty of Science and Technology, University of Twente, P.O. Box 217, 7500 AE Enschede, The Netherlands.
authors.forename: Richard B
authors.initials: RB
authors.lastname: Schasfoort
authors.affiliation: ‡Bioprocess Engineering, Wageningen University, P.O. Box 16, 6700 AA Wageningen, The Netherlands.|#University of Nordland, Faculty of Biosciences and Aquaculture, N-8049, Bodø, Norway.
authors.forename: René H
authors.initials: RH
authors.lastname: Wijffels
authors.affiliation: †Downstream Processing, Synthon Biopharmaceuticals BV, P.O. Box 7071, 6503 GN Nijmegen, The Netherlands.|‡Bioprocess Engineering, Wageningen University, P.O. Box 16, 6700 AA Wageningen, The Netherlands.
authors.forename: Michel H
authors.initials: MH
authors.lastname: Eppink
mesh_terms: D002623:Chemistry Techniques, Analytical / Q000379:methods*; D004921:Erythropoietin / Q000032:analysis* / Q000737:chemistry; D006031:Glycosylation; D037102:Lectins / Q000737:chemistry; D046228:Microarray Analysis; D011134:Polysaccharides / Q000737:chemistry*; D013194:Staining and Labeling; D020349:Surface Plasmon Resonance
chemical_list: D037102:Lectins; D011134:Polysaccharides; D004921:Erythropoietin