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.     

High-definition De Novo Sequencing of Crustacean Hyperglycemic Hormone (CHH)-family Neuropeptides

A complete understanding of the biological functions of large signaling peptides (>4 kDa) requires comprehensive characterization of their amino acid sequences and post-translational modifications, which presents significant analytical challenges. In the past decade, there has been great success with mass spectrometry-based de novo sequencing of small neuropeptides. However, these approaches are less applicable to larger neuropeptides because of the inefficient fragmentation of peptides larger than 4 kDa and their lower endogenous abundance. The conventional proteomics approach focuses on large-scale determination of protein identities via database searching, lacking the ability for in-depth elucidation of individual amino acid residues. Here, we present a multifaceted MS approach for identification and characterization of large crustacean hyperglycemic hormone (CHH)-family neuropeptides, a class of peptide hormones that play central roles in the regulation of many important physiological processes of crustaceans. Six crustacean CHH-family neuropeptides (8–9.5 kDa), including two novel peptides with extensive disulfide linkages and PTMs, were fully sequenced without reference to genomic databases. High-definition de novo sequencing was achieved by a combination of bottom-up, off-line top-down, and on-line top-down tandem MS methods. Statistical evaluation indicated that these methods provided complementary information for sequence interpretation and increased the local identification confidence of each amino acid. Further investigations by MALDI imaging MS mapped the spatial distribution and colocalization patterns of various CHH-family neuropeptides in the neuroendocrine organs, revealing that two CHH-subfamilies are involved in distinct signaling pathways.Neuropeptides and hormones comprise a diverse class of signaling molecules involved in numerous essential physiological processes, including analgesia, reward, food intake, learning and memory (1). Disorders of the neurosecretory and neuroendocrine systems influence many pathological processes. For example, obesity results from failure of energy homeostasis in association with endocrine alterations (2, 3). Previous work from our lab used crustaceans as model organisms found that multiple neuropeptides were implicated in control of food intake, including RFamides, tachykinin related peptides, RYamides, and pyrokinins (4–6).Crustacean hyperglycemic hormone (CHH)¹ family neuropeptides play a central role in energy homeostasis of crustaceans (7–17). Hyperglycemic response of the CHHs was first reported after injection of crude eyestalk extract in crustaceans. Based on their preprohormone organization, the CHH family can be grouped into two sub-families: subfamily-I containing CHH, and subfamily-II containing molt-inhibiting hormone (MIH) and mandibular organ-inhibiting hormone (MOIH). The preprohormones of the subfamily-I have a CHH precursor related peptide (CPRP) that is cleaved off during processing; and preprohormones of the subfamily-II lack the CPRP (9). Uncovering their physiological functions will provide new insights into neuroendocrine regulation of energy homeostasis.Characterization of CHH-family neuropeptides is challenging. They are comprised of more than 70 amino acids and often contain multiple post-translational modifications (PTMs) and complex disulfide bridge connections (7). In addition, physiological concentrations of these peptide hormones are typically below picomolar level, and most crustacean species do not have available genome and proteome databases to assist MS-based sequencing.MS-based neuropeptidomics provides a powerful tool for rapid discovery and analysis of a large number of endogenous peptides from the brain and the central nervous system. Our group and others have greatly expanded the peptidomes of many model organisms (3, 18–33). For example, we have discovered more than 200 neuropeptides with several neuropeptide families consisting of as many as 20–40 members in a simple crustacean model system (5, 6, 25–31, 34). However, a majority of these neuropeptides are small peptides with 5–15 amino acid residues long, leaving a gap of identifying larger signaling peptides from organisms without sequenced genome. The observed lack of larger size peptide hormones can be attributed to the lack of effective de novo sequencing strategies for neuropeptides larger than 4 kDa, which are inherently more difficult to fragment using conventional techniques (34–37). Although classical proteomics studies examine larger proteins, these tools are limited to identification based on database searching with one or more peptides matching without complete amino acid sequence coverage (36, 38).Large populations of neuropeptides from 4–10 kDa exist in the nervous systems of both vertebrates and invertebrates (9, 39, 40). Understanding their functional roles requires sufficient molecular knowledge and a unique analytical approach. Therefore, developing effective and reliable methods for de novo sequencing of large neuropeptides at the individual amino acid residue level is an urgent gap to fill in neurobiology. In this study, we present a multifaceted MS strategy aimed at high-definition de novo sequencing and comprehensive characterization of the CHH-family neuropeptides in crustacean central nervous system. The high-definition de novo sequencing was achieved by a combination of three methods: (1) enzymatic digestion and LC-tandem mass spectrometry (MS/MS) bottom-up analysis to generate detailed sequences of proteolytic peptides; (2) off-line LC fractionation and subsequent top-down MS/MS to obtain high-quality fragmentation maps of intact peptides; and (3) on-line LC coupled to top-down MS/MS to allow rapid sequence analysis of low abundance peptides. Combining the three methods overcomes the limitations of each, and thus offers complementary and high-confidence determination of amino acid residues. We report the complete sequence analysis of six CHH-family neuropeptides including the discovery of two novel peptides. With the accurate molecular information, MALDI imaging and ion mobility MS were conducted for the first time to explore their anatomical distribution and biochemical properties.