title: Ultrahigh throughput, ultrafiltration-based n-glycomics platform for ultraperformance liquid chromatography (ULTRA(3)).
abstract: Accurate, reproducible, and fast quantification of N-glycans is crucial not only for the development and quality control of modern glycosylated biopharmaceuticals, but also in clinical biomarker discovery. Several methods exist for fluorescent labeling of N-glycans and subsequent chromatographic separation and quantification. However, the methods can be complex, lengthy, and expensive. Here we report an automated ultrafiltration-based N-glycoanalytical workflow combined with a glycan labeling strategy that is based on the reaction of glycosylamines with fluorescent carbamate. The entire protocol is quick, simple, and cost-effective and requires less than 1 μg of protein per sample. As many as 768 affinity purified IgG glycoprotein samples can be prepared in a single run with a liquid handling platform.
authors.affiliation: NIBRT GlycoScience Group, NIBRT - The National Institute for Bioprocessing Research and Training, Fosters Avenue, Mount Merrion, Blackrock, Co. Dublin, Ireland.
authors.forename: Henning
authors.initials: H
authors.lastname: Stöckmann
authors.affiliation: NIBRT GlycoScience Group, NIBRT - The National Institute for Bioprocessing Research and Training, Fosters Avenue, Mount Merrion, Blackrock, Co. Dublin, Ireland.
authors.forename: Rebecca M
authors.initials: RM
authors.lastname: Duke
authors.affiliation: NIBRT GlycoScience Group, NIBRT - The National Institute for Bioprocessing Research and Training, Fosters Avenue, Mount Merrion, Blackrock, Co. Dublin, Ireland.
authors.forename: Silvia
authors.initials: S
authors.lastname: Millán Martín
authors.affiliation: NIBRT GlycoScience Group, NIBRT - The National Institute for Bioprocessing Research and Training, Fosters Avenue, Mount Merrion, Blackrock, Co. Dublin, Ireland.
authors.forename: Pauline M
authors.initials: PM
authors.lastname: Rudd
mesh_terms: D000634:Aminoquinolines / Q000737:chemistry*; D002219:Carbamates / Q000737:chemistry*; D002623:Chemistry Techniques, Analytical / Q000295:instrumentation* / Q000379:methods*; D002853:Chromatography, Liquid / Q000295:instrumentation*; D005456:Fluorescent Dyes / Q000737:chemistry; D054794:Glycomics; D011134:Polysaccharides / Q000737:chemistry; D014462:Ultrafiltration
chemical_list: C078178:6-aminoquinolyl-N-hydroxysuccinimidyl carbamate; D000634:Aminoquinolines; D002219:Carbamates; D005456:Fluorescent Dyes; D011134:Polysaccharides