title: Comparative Analysis and Validation of Different Steps in Glycomics Studies.
abstract: Large-scale glycomics studies enable identification of aberrant glycosylation patterns in disease and provide information about functional relevance of individual glycans through genome-wide association studies. Developed high-throughput methodologies have to be sensitive, robust, and stable during long periods of time (few months) to be able to reliably detect small biological variations in glycosylation. Here, we describe a simple, robust, and affordable protocol for immunoglobulin G N-glycan analysis by hydrophilic interaction liquid chromatography-ultra-performance liquid chromatography (HILIC-UPLC), as well as useful strategies for method optimization: Plackett-Burman screening design and analysis of source of variation. We put our focus on experimental design for high-throughput glycan analysis, critical steps in sample preparation procedure for obtaining high-quality data, and propose a validation protocol relevant for high-throughput methods in terms of their long-term robustness and ability to detect biologically relevant changes in glycosylation. The quality of the procedure was assessed by employing appropriate experimental designs and subsequent statistical techniques.
authors.affiliation: Genos Glycoscience Research Laboratory, Zagreb, Croatia.
authors.forename: I
authors.initials: I
authors.lastname: Trbojević-Akmačić
authors.affiliation: Genos Glycoscience Research Laboratory, Zagreb, Croatia; Faculty of Pharmacy and Biochemistry, University of Zagreb, Zagreb, Croatia.
authors.forename: I
authors.initials: I
authors.lastname: Ugrina
authors.affiliation: Genos Glycoscience Research Laboratory, Zagreb, Croatia; Faculty of Pharmacy and Biochemistry, University of Zagreb, Zagreb, Croatia. Electronic address: glauc@genos.hr.
authors.forename: G
authors.initials: G
authors.lastname: Lauc
mesh_terms: D002851:Chromatography, High Pressure Liquid; D005260:Female; D054794:Glycomics / Q000379:methods; D006031:Glycosylation; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D007074:Immunoglobulin G / Q000737:chemistry / Q000302:isolation & purification*; D008297:Male; D011499:Protein Processing, Post-Translational
chemical_list: D007074:Immunoglobulin G
keywords: Glycans; Glycome analysis; HILIC; High throughput; Method validation; UPLC