title: Assignment of Core versus Antenna Fucosylation Types in Protein N-Glycosylation via Procainamide Labeling and Tandem Mass Spectrometry.
abstract: Fucosylation is an important feature of protein N-glycosylation as it has been reported to influence the efficacy of therapeutic proteins and as a potential disease biomarker. A common approach for characterizing protein N-glycans is to analyze the native glycans via tandem mass spectrometry (MS). However, tandem MS analysis of native N-glycans typically results in proton migration, which in turn leads to fucose residue migration from the glycan core to the antenna and vice versa. This phenomenon ultimately leads to ambiguous assignment of N-glycan fucosylation. Although the use of specific fucosidases has been successfully employed for assigning fucosylation, such strategies are often too cumbersome, expensive, and time-consuming for routine N-glycan analysis. As an alternative, we explore the influence of labeling N-glycans with procainamide hydrochloride to inhibit fucose migration during tandem MS analysis. The labeled N-glycan pool was separated and analyzed using ultraperformance liquid chromatography and a hydrophobic interaction liquid chromatography column coupled to a quadrupole time-of-flight mass spectrometer (UPLC-HILIC-QTOF-MS). The observation of the m/z 587.3 core fucose diagnostic peak corresponding to [GlcNAc + Fucose + Procainamide + H](+) in the tandem MS data of fucosylated N-glycans rapidly verifies core fucosylation while its absence signifies antennae fucosylation. This unique approach is here validated with human IgG (for core fucosylation) and human alpha-1-acid-glycoprotein (for antenna fucosylation). We further present a useful application toward the rapid verification of fucosylation types in a therapeutic protein (Rituximab).
authors.affiliation: Pharmaceutical Product Development, 8551 Research Way, Middleton, Wisconsin 53562, United States.
authors.forename: Charles
authors.initials: C
authors.lastname: Nwosu
authors.affiliation: Pharmaceutical Product Development, 8551 Research Way, Middleton, Wisconsin 53562, United States.
authors.forename: Hoi Kei
authors.initials: HK
authors.lastname: Yau
authors.affiliation: Pharmaceutical Product Development, 8551 Research Way, Middleton, Wisconsin 53562, United States.
authors.forename: Steven
authors.initials: S
authors.lastname: Becht
mesh_terms: D005643:Fucose / Q000737:chemistry; D006031:Glycosylation; D006801:Humans; D007074:Immunoglobulin G / Q000737:chemistry*; D009961:Orosomucoid / Q000737:chemistry*; D011134:Polysaccharides / Q000032:analysis*; D011342:Procainamide / Q000737:chemistry*; D013194:Staining and Labeling; D053719:Tandem Mass Spectrometry
chemical_list: D007074:Immunoglobulin G; D009961:Orosomucoid; D011134:Polysaccharides; D005643:Fucose; D011342:Procainamide