Comparative Performance of Four Methods for High-throughput Glycosylation Analysis of Immunoglobulin G in Genetic and Epidemiological Research

The biological and clinical relevance of glycosylation is becoming increasingly recognized, leading to a growing interest in large-scale clinical and population-based studies. In the past few years, several methods for high-throughput analysis of glycans have been developed, but thorough validation and standardization of these methods is required before significant resources are invested in large-scale studies. In this study, we compared liquid chromatography, capillary gel electrophoresis, and two MS methods for quantitative profiling of N-glycosylation of IgG in the same data set of 1201 individuals. To evaluate the accuracy of the four methods we then performed analysis of association with genetic polymorphisms and age. Chromatographic methods with either fluorescent or MS-detection yielded slightly stronger associations than MS-only and multiplexed capillary gel electrophoresis, but at the expense of lower levels of throughput. Advantages and disadvantages of each method were identified, which should inform the selection of the most appropriate method in future studies.Glycans are important structural and functional components of the majority of proteins, but because of their structural complexity and the absence of a direct genetic template our current understanding of the role of glycans in biological processes lags significantly behind the knowledge about proteins or DNA (1, 2). However, a recent comprehensive report endorsed by the US National Academies concluded that “glycans are directly involved in the pathophysiology of every major disease and that additional knowledge from glycoscience will be needed to realize the goals of personalized medicine” (3).It is estimated that the glycome (defined as the complete set of all glycans) of a eukaryotic cell is composed of more than a million different glycosylated structures (1), which contain up to 10,000 structural glycan epitopes for interaction with antibodies, lectins, receptors, toxins, microbial adhesins, or enzymes (4). Our recent population-based studies indicated that the composition of the human plasma N-glycome varies significantly between individuals (5, 6). Because glycans have important structural and regulatory functions on numerous glycoproteins (7), the observed variability suggests that differences in glycosylation might contribute to a large part of the human phenotypic variability. Interestingly, when the N-glycome of isolated immunoglobulin G (IgG)¹ was analyzed, it was found to be even more variable than the total plasma N-glycome (8), indicating that the combined analysis of all plasma glycans released from many different glycoproteins blurs signals of protein-specific regulation of glycosylation.A number of studies have investigated the role of glycans in human disease, including autoimmune diseases and cancer (9, 10). However, most human glycan studies have been conducted with very small sample sizes. Given the complex causal pathways involved in pathophysiology of common complex disease, and thus the likely modest effect sizes associated with individual factors, the majority of these studies are very likely to be substantially underpowered. In the case of inflammatory bowel disease, only 20% of reported inflammatory bowel disease glycan associations were replicated in subsequent studies, suggesting that most are false positive findings and that there is publication bias favoring the publication of positive findings (11). This situation is similar to that which occurred in the field of genetic epidemiology in the past when many underpowered candidate gene studies were published and were later found to consist of mainly false positive findings (12, 13). It is essential, therefore, that robust and affordable methods for high-throughput analysis are developed so that adequately powered studies can be conducted and the publication of large numbers of small studies reporting false positive results (which could threaten the credibility of glycoscience) be avoided.Rapid advances of technologies for high-throughput genome analysis in the past decade enabled large-scale genome-wide association studies (GWAS). GWAS has become a reliable tool for identification of associations between genetic polymorphisms and various human diseases and traits (14). Thousands of GWAS have been conducted in recent years, but these have not included the study of glycan traits until recently. The main reason was the absence of reliable tools for high-throughput quantitative analysis of glycans that could match the measurements of genomic, biochemical, and other traits in their cost, precision, and reproducibility. However, several promising high-throughput technologies for analysis of N-glycans were developed (8, 15–20) recently. Successful implementation of high-throughput analytical techniques for glycan analysis resulted in publication of four initial GWAS of the human glycome (21–24).In this study, we compared ultra-performance liquid chromatography with fluorescence detection (UPLC-FLR), multiplex capillary gel electrophoresis with laser induced fluorescence detection (xCGE-LIF), matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS), and liquid chromatography electrospray mass spectrometry (LC-ESI-MS) as tools for mid-to-high-throughput glycomics and glycoproteomics. We have analyzed IgG N-glycans by all four methods in 1201 individuals from European populations. The analysis of associations between glycans and ∼300,000 single-nucleotide genetic polymorphisms was performed and correlation between glycans and age was studied in all four data sets to identify the analytical method that shows the strongest potential to uncover biological mechanisms underlying protein glycosylation.