Comparative Analysis and Validation of Different Steps in Glycomics Studies.

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.