High-Throughput Analysis of the IgG N-Glycome by UPLC-FLR.

As biological and clinical relevance of glycosylation is becoming more apparent, interest in large scale studies of the glycome is growing. Glycans attached to immunoglobulin G (IgG) were shown to be essential for its function and IgG glycosylation was shown to change with various processes, making IgG one of the most studied glycoproteins. Many approaches including liquid chromatography, capillary gel electrophoresis, and mass spectrometry were developed to study IgG glycosylation. Generation of high-quality glycomics data in a high-throughput fashion requires reproducible and robust sample preparation and accurate and reliable quantitative analysis. This chapter presents a protocol for an optimized and high-throughput IgG N-glycan release, fluorescent labeling and cleanup, and analysis of fluorescently labeled IgG N-glycans by hydrophilic interaction liquid chromatography (HILIC) on an ultra performance liquid chromatography (UPLC) system with fluorescence (FLR) detection.