title: Detection of Protein Glycosylation Using Tip-Enhanced Raman Scattering.
abstract: The correct glycosylation of biopharmaceutical glycoproteins and their formulations is essential for them to have the desired therapeutic effect on the patient. It has recently been shown that Raman spectroscopy can be used to quantify the proportion of glycosylated protein from mixtures of native and glycosylated forms of bovine pancreatic ribonuclease (RNase). Here we show the first steps toward not only the detection of glycosylation status but the characterization of glycans themselves from just a few protein molecules at a time using tip-enhanced Raman scattering (TERS). While this technique generates complex data that are very dependent on the protein orientation, with the careful development of combined data preprocessing, univariate and multivariate analysis techniques, we have shown that we can distinguish between the native and glycosylated forms of RNase. Many glycoproteins contain populations of subtly different glycoforms; therefore, with stricter orientation control, we believe this has the potential to lead to further glycan characterization using TERS, which would have use in biopharmaceutical synthesis and formulation research.
authors.affiliation: School of Chemistry and Manchester Institute of Biotechnology, University of Manchester , 131 Princess Street, Manchester, M1 7DN, U.K.
authors.forename: David P
authors.initials: DP
authors.lastname: Cowcher
authors.affiliation: Leibniz-Institute of Photonic Technology-IPHT , Albert-Einstein-Strasse 9, 07745 Jena, Germany.
authors.forename: Tanja
authors.initials: T
authors.lastname: Deckert-Gaudig
authors.affiliation: School of Chemistry and Manchester Institute of Biotechnology, University of Manchester , 131 Princess Street, Manchester, M1 7DN, U.K.
authors.forename: Victoria L
authors.initials: VL
authors.lastname: Brewster
authors.affiliation: School of Chemistry and Manchester Institute of Biotechnology, University of Manchester , 131 Princess Street, Manchester, M1 7DN, U.K.|Department of Chemistry, Faraday Building, Lancaster University , Lancaster, LA1 4YB, U.K.
authors.forename: Lorna
authors.initials: L
authors.lastname: Ashton
authors.affiliation: Leibniz-Institute of Photonic Technology-IPHT , Albert-Einstein-Strasse 9, 07745 Jena, Germany.|Institut für Physikalische Chemie and Abbe Center of Photonics, Friedrich-Schiller Universität , Helmholtzweg 4, 07743 Jena, Germany.
authors.forename: Volker
authors.initials: V
authors.lastname: Deckert
authors.affiliation: School of Chemistry and Manchester Institute of Biotechnology, University of Manchester , 131 Princess Street, Manchester, M1 7DN, U.K.
authors.forename: Royston
authors.initials: R
authors.lastname: Goodacre
mesh_terms: D000818:Animals; D002417:Cattle; D006031:Glycosylation; D006046:Gold / Q000737:chemistry; D016018:Least-Squares Analysis; D018625:Microscopy, Atomic Force; D015999:Multivariate Analysis; D049329:Nanostructures / Q000737:chemistry; D025341:Principal Component Analysis; D012259:Ribonuclease, Pancreatic / Q000032:analysis* / Q000378:metabolism; D013059:Spectrum Analysis, Raman
chemical_list: D006046:Gold; D012259:Ribonuclease, Pancreatic