Search

Ultra-performance liquid chromatography (UPLC) is the established technology for accurate analysis of IgG Fc N-glycosylation due to its superior sensitivity, resolution, speed, and its capability to provide branch-specific information of glycan species. Correct and cost-efficient preprocessing of chromatographic data is the major prerequisite for subsequent analyses ranging from inference of structural isomers to biomarker discovery and prediction of humoral immune response from characterized changes in glycosylation. The complexity of glycomic chromatograms poses a number of challenges for developing automated data annotation and quantitation algorithms, which frequently necessitated manual or semi-manual approaches to preprocessing, most notably to peak detection and integration. Such procedures are meticulous and time-consuming, and may be a source of confounding due to their dependence on human labelers. Although liquid chromatography is a mature field and a number of methods have been developed for automatic peak detection outside the area of glycomics analysis, we found that hardly any of them are suitable for automatic integration of UPLC glycomic profiles without substantial modifications. In this chapter, we illustrate practical challenges of automatic peak detection of UPLC glycomics chromatograms. We outline a robust, semi-supervised method ACE (Automatic Chromatogram Extraction) for automated alignment and detection of glycan peaks in chromatograms, developed by Pharmatics Limited (UK) in collaboration with Genos Limited (Croatia). Application of the tool requires minimal human interference, which results in a significant reduction in the time and cost of IgG glycomics signal integration using Waters Acquity UPLC instrument (Milford, MA, USA) in several human cohorts with blind technical replicas.

Post-translational modifications diversify protein functions and dynamically coordinate their signalling networks, influencing most aspects of cell physiology. Nevertheless, their genetic regulation or influence on complex traits is not fully understood. Here, we compare the genetic regulation of the same PTM of two proteins – glycosylation of transferrin and immunoglobulin G (IgG). By performing genome-wide association analysis of transferrin glycosylation, we identify 10 significantly associated loci, 9 of which were not reported previously. Comparing these with IgG glycosylation-associated genes, we note protein-specific associations with genes encoding glycosylation enzymes (transferrin - MGAT5, ST3GAL4, B3GAT1; IgG - MGAT3, ST6GAL1), as well as shared associations (FUT6, FUT8). Colocalisation analyses of the latter suggest that different causal variants in the FUT genes regulate fucosylation of the two proteins. Glycosylation of these proteins is thus genetically regulated by both shared and protein-specific mechanisms.