Methods for the absolute quantification of N-glycan biomarkers

BackgroundMany treatment options especially for cancer show a low efficacy for the majority of patients demanding improved biomarker panels for patient stratification. Changes in glycosylation are a hallmark of many cancers and inflammatory diseases and show great potential as clinical disease markers. The large inter-subject variability in glycosylation due to hereditary and environmental factors can complicate rapid transfer of glycan markers into the clinical practice but also presents an opportunity for personalized medicine.Scope of reviewThis review discusses opportunities of glycan biomarkers in personalized medicine and reviews the methodology for N-glycan analysis with a specific focus on methods for absolute quantification.Major conclusionsThe entry into the clinical practice of glycan markers is delayed in large part due to a lack of adequate methodology for the precise and robust quantification of protein glycosylation. Only absolute glycan quantification can provide a complete picture of the disease related changes and will provide the method robustness required by clinical applications.General significanceGlycan biomarkers have a huge potential as disease markers for personalized medicine. The use of stable isotope labeled glycans as internal standards and heavy-isotope labeling methods will provide the necessary method precision and robustness acceptable for clinical use. This article is part of a Special Issue entitled “Glycans in personalized medicine” Guest Editor: Professor Gordan Lauc.     

Clinical application of quantitative glycomics

ABSTRACT

Introduction: Aberrant glycosylation has been associated with many diseases. Decades of research activities have reported many reliable glycan biomarkers of different diseases which enable effective disease diagnostics and prognostics. However, none of the glycan markers have been approved for clinical diagnosis. Thus, a review of these studies is needed to guide the successful clinical translation.

Area covered: In this review, we describe and discuss advances in analytical methods enabling clinical glycan biomarker discovery, focusing only on studies of released glycans. This review also summarizes the different glycobiomarkers identified for cancers, Alzheimer’s disease, diabetes, hepatitis B and C, and other diseases.

Expert commentary: Along with the development of techniques in quantitative glycomics, more glycans or glycan patterns have been reported as better potential biomarkers of different diseases and proved to have greater diagnostic/diagnostic sensitivity and specificity than existing markers. However, to successfully apply glycan markers in clinical diagnosis, more studies and verifications on large biological cohorts need to be performed. In addition, faster and more efficient glycomic strategies need to be developed to shorten the turnaround time. Thus, glycan biomarkers have an immense chance to be used in clinical prognosis and diagnosis of many diseases in the near future.     

Glycomics and glycoproteomics focused on aging and age-related diseases — Glycans as a potential biomarker for physiological alterations

BackgroundSince glycosylation depends on glycosyltransferases, glycosidases, and sugar nucleotide donors, it is susceptible to the changes associated with physiological and pathological conditions. Therefore, alterations in glycan structures may be good targets and biomarkers for monitoring health conditions. Since human aging and longevity are affected by genetic and environmental factors such as diseases, lifestyle, and social factors, a scale that reflects various environmental factors is required in the study of human aging and longevity.Scope of reviewWe herein focus on glycosylation changes elucidated by glycomic and glycoproteomic studies on aging, longevity, and age-related diseases including cognitive impairment, diabetes mellitus, and frailty. We also consider the potential of glycan structures as biomarkers and/or targets for monitoring physiological and pathophysiological changes.Major conclusionsGlycan structures are altered in age-related diseases. These glycans and glycoproteins may be involved in the pathophysiology of these diseases and, thus, be useful diagnostic markers. Age-dependent changes in N-glycans have been reported previously in cohort studies, and characteristic N-glycans in extreme longevity have been proposed. These findings may lead to a deeper understanding of the mechanisms underlying aging as well as the factors influencing longevity.General significanceAlterations in glycosylation may be good targets and biomarkers for monitoring health conditions, and be applicable to studies on age-related diseases and healthy aging. This article is part of a Special Issue entitled "Glycans in personalised medicine" Guest Editor: Professor Gordan Lauc.     

The promise of protein glycosylation for personalised medicine

BackgroundComplex diseases such as cancer are a consequence of numerous causes. State of the art personalised medicine approaches are mostly based on evaluating patients' individual genetic background. Despite the advances of genomics it fails to take individual dynamic influences into account that contribute to the individual and unique glycomic and glycoproteomic “configurations” of every living being.Scope of reviewGlycomic and glycoproteomic-based personalised medicine diagnostics are still in their infancies, however some initial success stories indicate that these fields are highly promising to mediate novel early diagnosis and disease stratification markers, subsequently resulting in improved patient well-being and reduced treatment costs. In this review we not only summarise current protein glycosylation based examples that substantially improve or possess great potential for personalised medicine, but also describe current limitations as well as future perspectives and challenges associated with establishing protein glycosylation aspects for this purpose.Major conclusionsMany protein biomarkers currently in clinical use are glycoproteins, however, their glycosylation status is seldom evaluated in a clinical context. To date just few examples have already been successfully translated into clinical practice, making protein glycosylation a highly promising diagnostic target with humongous potential for personalised medicine.General significanceThere is an urgent need for markers that enable the establishment of an individualised and optimised patient treatment at the earliest disease stage possible. The glycosylation status of a patient and/or specific marker proteins can provide important clues that result in improved patient management. This article is part of a Special Issue entitled “Glycans in personalised medicine” Guest Editor: Professor Gordan Lauc.     

The Potentials of Glycomics in Biomarker Discovery

Introduction

Glycans have unique characteristics that are significantly different from nucleic acids and proteins in terms of biosynthesis, structures, and functions. Moreover, their isomeric nature and the complex linkages between residues have made glycan analysis a challenging task. Disease development and progression are usually associated with alternations in glycosylation on tissue proteins and/or blood proteins. Glycans released from tissue/blood proteins hence provide a valuable source of biomarkers. In this postgenome era, glycomics is an emerging research field. Glycome refers to a repertoire of glycans in a tissue/cell type, while glycomics is the study of glycome. In the past few years, attempts have been made to develop novel methodologies for quantitative glycomic profiling and to identify potential glycobiomarkers. It can be foreseen that glycomics holds the promise for biomarker discovery. This review provides an overview of the unique features of glycans and the historical applications of such features to biomarker discovery.

Future Prospective

The concept of glycomics and its recent advancement and future prospective in biomarker research are reviewed. Above all, there is no doubt that glycomics is gaining momentum in biomarker research.     

Glycosylation in viral hepatitis

BackgroundThe interaction between hepatitis viruses and host cells is regulated by glycans exposed on the surfaces of human and viruses cells. As the biosynthesis and degradation of human glycoproteins take place at the highest level in the liver, the changes in glycosylation of serum proteins may potentially be useful in the diagnosis of liver pathology. On the other hand, specific alterations in viruses envelope glycans could cause large changes in the entry process of hepatitis viruses into a host cells.Scope of reviewUnique alterations in glycosylation of specific proteins can be detected in HBV and HCV infected patients especially with confirmed fibrosis/cirrhosis. On the other hand, viral envelope proteins that bind to host cells are glycosylated. These glycosylated proteins play a key role in recognition, binding and penetration of the host cells. In this review we summarized the knowledge about significance of glycosylation for viral and host factors.Major conclusionsGlycosylation changes in single serum glycoproteins are noticed in the sera of patients with viral hepatitis. However, a more specific biomarker for the diagnosis of chronic hepatitis than that of a single glycosylated molecule is systemic investigation of complete set of glycan structures (N-glycome). Glycans play important roles in the viral biology cycle especially as a connecting element with host receptors.General significanceThe interaction between virus glycoproteins and cellular receptors, which are also glycoproteins, determines the possibility of virus penetration into host cells. Therefore these glycans can be the targets for the developing of novel treatment strategies of viral hepatitis.     

IgG and IgM glycosylation patterns in patients undergoing image-guided tumor ablation

BackgroundImage-guided tumor ablation is a technique whereby needle-like applicators are placed directly into solid tumors under guidance typically with computed tomography or ultrasound. Changes in IgG and IgM antibody glycosylation were studied during ablation-induced immune response to cancer, and the use of glycosylation as a biomarker for diagnosis, prognosis and disease treatment was examined.MethodsPlasma from 27 tumor patients was collected immediately before, after and for 6 months following ablation. IgG and IgM antibodies were isolated by use high-throughput chromatography, and analyzed by hydrophilic liquid chromatography. Thorough identification of glycan structures in each chromatography peak was performed by nano-liquid chromatography electrospray ionization mass spectrometry.ResultsAlthough antibody glycosylation was found to vary with cancer type, discernable patterns of change based on the successful treatment of tumors by ablation were not identified. One patient with renal clear cell carcinoma and poor disease outcome had unexpectedly high amount of oligomannose IgG glycans during the whole period of monitoring. In contrast, IgM antibodies did not follow the same pattern.ConclusionsThese findings suggest that glycosylation patterns are indicative of an immune system that is unable to prevent different types of cancer, rather than products of the immunostimulatory response to the ablation of tumor itself. Analyses of the outcome effect suggested that IgG glycosylation and IgM glycosylation are not associated with tumor ablation.General significancePresent work opens a new way for parallel determination of glycosylation changes of both IgG and IgM antibodies by use of high-throughput methods, and their future use as biomarkers for disease diagnosis and prognosis. This article is part of a Special Issue entitled "Glycans in personalised medicine" Guest Editor: Professor Gordan Lauc.     

Glyco-genes change expression in cancer through aberrant methylation

BackgroundMost eukaryotic proteins are modified by covalent addition of glycan molecules that considerably influence their function. Aberrant glycosylation is profoundly involved in malignant transformation, tumor progression and metastasis. Some glycan structures are tumor-specific and reflect disturbed glycan biosynthesis pathways.MethodsWe analyzed DNA methylation and expression of 86 glyco-genes in melanoma, hepatocellular, breast and cervical cancers using data from publicly available databases. We also analyzed methylation datasets without the available matching expression data for glyco-genes in lung cancer, and progression of melanoma into lymph node and brain metastases.ResultsTen glyco-genes (GALNT3, GALNT6, GALNT7, GALNT14, MGAT3, MAN1A1, MAN1C1, ST3GAL2, ST6GAL1, ST8SIA3) showing changes in both methylation and expression in the same type of cancer belong to GalNAc transferases, GlcNAc transferases, mannosidases and sialyltransferases, which is in line with changes in glycan structures already reported in the same type of tumors. Some of those genes were additionally identified as potentially valuable for disease prognosis. The MGAT5B gene, so far identified as specifically expressed in brain, emerged as a novel candidate gene that is epigenetically dysregulated in different cancers other than brain cancer. We also report for the first time aberrant expression of the GALNT and MAN genes in cancer by aberrant promoter methylation.ConclusionsAberrant expression of glyco-genes due to aberrant promoter methylation could be a way leading to characteristic glycosylation profiles commonly described in cancer.General significanceMethylation status in promoters of candidate glyco-genes might serve as prognostic markers for specific tumors and point to potential novel targets for epigenetic drugs.This article is part of a Special Issue entitled "Glycans in personalised medicine" Guest Editor: Professor Gordan Lauc.     

N-glycan and Alzheimer's disease

BackgroundAlzheimer's disease (AD) is a major form of dementia. Many evidence-based clinical trials have been performed, but no effective treatment has yet been developed. This suggests that our understanding of AD patho-mechanisms is still insufficient. In particular, the pathological roles of posttranslational modifications including glycosylation have remained poorly understood, but recent advances in glycobiology technology have gradually revealed that sugar modifications of AD-related molecules are profoundly involved in the onset and progression of this disease.Scope of reviewWe summarize the roles of N-glycans in AD pathogenesis and progression, particularly focusing on key AD-related molecules, including amyloid precursor protein (APP), α-, β-, and γ-secretases, and tau.Major conclusionsBiochemical, genetic and pharmacological studies have gradually revealed how N-glycans regulate AD development and progression through functional modulation of the key glycoproteins. These findings suggest that further glycobiology approaches in AD research will reveal novel glycan-based drug targets and early biomarkers of AD. However, N-glycan structures of these molecules in physiological and disease conditions and their precise functions are still largely unclear. Deeper glycobiology studies will be needed to reveal how AD pathology is regulated by glycosylation.General significanceIt is now known that N-glycans play significant roles in AD development. However, specific pathological functions of particular glycan epitopes on each AD-related glycoprotein are still poorly understood. Future glycobiology studies with more sensitive glycoproteomic techniques and a wider variety of chemical glycosylation inhibitors could contribute to the development of novel glycan-based AD therapeutics. This article is part of a Special Issue entitled Neuro-glycoscience, edited by Kenji Kadomatsu and Hiroshi Kitagawa.     

Liquid Chromatography/Mass Spectrometry (LC/MS)-Based Glycoproteomics Technologies for Cancer Biomarker Discovery

Introduction

Biomarker discovery is a major objective of clinical proteomics; molecular biomarkers allow for detection of early-stage human diseases, especially cancer, and for monitoring their progression and/or regression after treatment. Biomarkers also help to elucidate the pathology of disease and its diagnosis, drug discovery, and toxicology. Glycans are ideal candidates for biomarkers because (1) glycoconjugates are localized on the cell surface and in the secretions such as plasma, (2) their structures are frequently and drastically changed during normal and aberrant cell differentiation, and (3) different cell types express different glycan signatures. Certain serodiagnostic glycoconjugate markers, such as carcinoembryonic antigen (CEA), are currently available; however, comprehensive glycome analysis has yet to be performed, mainly because of the difficulties of isolating and structurally analyzing complex glycans. Large-scale glycoprotein analysis, termed glycoproteomics, has the potential to effectively trace cellular glycoproteins and therefore to search for new serodiagnostic biomarkers.

Conclusions

In this review, we describe current mass spectrometry-based glycoproteomics technologies. Quantitative “shotgun” proteomics analyses of glycopeptides captured from complex biological mixtures such as plasma, coupled with advanced glycome technologies, enhance our knowledge of protein glycosylation and facilitate discovery of new biomarkers for human diseases.     

Glycoblotting method allows for rapid and efficient glycome profiling of human Alzheimer's disease brain,serum and cerebrospinal fluid towards potential biomarker discovery

BackgroundUnderstanding of the significance of posttranslational glycosylation in Alzheimer's disease (AD) is of growing importance for the investigation of the pathogenesis of AD as well as discovery research of the disease-specific serum biomarkers.MethodsWe designed a standard protocol for the glycoblotting combined with MALDI-TOFMS to perform rapid and quantitative profiling of the glycan parts of glycoproteins (N-glycans) and glycosphingolipids (GSLs) using human AD's post-mortem samples such as brain tissues (dissected cerebral cortices such as frontal, parietal, occipital, and temporal domains), serum and cerebrospinal fluid (CSF).ResultsThe structural profiles of the major N-glycans released from glycoproteins and the total expression levels of the glycans were found to be mostly similar between the brain tissues of the AD patients and those of the normal control group. In contrast, the expression levels of the serum and CSF protein N-glycans such as bisect-type and multiply branched glycoforms were increased significantly in AD patient group. In addition, the levels of some gangliosides such as GM1, GM2 and GM3 appeared to alter in the AD patient brain and serum samples when compared with the normal control groups.ConclusionAlteration of the expression levels of major N- and GSL-glycans in human brain tissues, serum and CSF of AD patients can be monitored quantitatively by means of the glycoblotting-based standard protocols.General significanceThe changes in the expression levels of the glycans derived from the human post-mortem samples uncovered by the standardized glycoblotting method provides potential serum biomarkers in central nervous system disorders and can contribute to the insight into the molecular mechanisms in the pathogenesis of neurodegenerative diseases and future drug discovery. Most importantly, the present preliminary trials using human post-mortem samples of AD patients suggest that large-scale serum glycomics cohort by means of various-types of human AD patients as well as the normal control sera can facilitate the discovery research of highly sensitive and reliable serum biomarkers for an early diagnosis of AD. This article is part of a Special Issue entitled "Glycans in personalised medicine" Guest Editor: Professor Gordan Lauc.     

High-throughput analysis of immunoglobulin G glycosylation

Introduction: Glycosylation of immunoglobulin G (IgG) is important for its effector functions and was shown to be related to age, sex and disease status of an individual. Adding glycomic information to genome-wide association studies (GWAS) and large clinical trials is enabling insight into the functional relevance of changes in glycosylation, as well as molecular mechanisms behind these changes. Large-scale studies require sensitive, robust and affordable high-throughput methodologies for glycosylation analysis, which are currently available in only a limited number of laboratories.

Areas covered: This review focuses on currently used high-throughput approaches for N-glycosylation analysis of IgG, as well as some recent advances in the areas of deglycosylation, trypsin digestion, labeling, purification, derivatization and automation of current workflows. Relevant literature was searched using the PubMed database.

Expert commentary: Development, optimization and validation of robust, affordable and simple high-throughput glycosylation analysis methods is essential for discovery and validation of diagnostic and prognostic glycan biomarkers. Although significant advances in glycosylation analysis have been made in recent years, currently used protocols will have to be further optimized to enable subsequent analysis of glycosylation on all levels with the limited initial sample and in the minimal amount of time, which is still a challenging task.     

The use of lectin microarray for assessing glycosylation of therapeutic proteins

Glycans or carbohydrates attached to therapeutic glycoproteins can directly affect product quality, safety and efficacy, and therefore must be adequately analyzed and controlled throughout product life cycles. However, the complexity of protein glycosylation poses a daunting analytical challenge. In this study, we evaluated the utility of a lectin microarray for assessing protein glycans. Using commercial lectin chips, which contain 45 lectins toward distinct glycan structures, we were able to determine the lectin binding patterns of a panel of 15 therapeutic proteins, including 8 monoclonal antibodies. Lectin binding signals were analyzed to generate glycan profiles that were generally consistent with the known glycan patterns for these glycoproteins. In particular, the lectin-based microarray was found to be highly sensitive to variations in the terminal carbohydrate structures such as galactose versus sialic acid epitopes. These data suggest that lectin microarray could be used for screening glycan patterns of therapeutic glycoproteins.     

Glycobiomarkers by glycoproteomics and glycan profiling (glycomics): emergence of functionality

Glycans stand out from all classes of biomolecules because of their unsurpassed structural complexity. This is generated by variability in anomeric status of the glycosidic bond and its linkage points, ring size, potential for branching and introduction of diverse site-specific substitutions. What poses an enormous challenge for analytical processing is, at the same time, the basis for the fingerprint-like glycomic profiles of glycoconjugates and cells. What's more, the glycosylation machinery is sensitive to disease manifestations, earning glycan assembly a reputation as a promising candidate to identify new biomarkers. Backing this claim for a perspective in clinical practice are recent discoveries that even seemingly subtle changes in the glycan structure of glycoproteins, such as a N-glycan core substitution by a single sugar moiety, have far-reaching functional consequences. They are brought about by altering the interplay between the glycan and (i) its carrier protein and (ii) specific receptors (lectins). Glycan attachment thus endows the protein with a molecular switch and new recognition sites. Co-ordinated regulation of glycan display and presentation of the cognate lectin, e.g. in cancer growth regulation exerted by a tumour suppressor, further exemplifies the broad functional dimension inherent to the non-random shifts in glycosylation. Thus studies on glycobiomarkers converge with research on how distinct carbohydrate determinants are turned into bioactive signals.     

Inflammatory bowel disease - glycomics perspective

BackgroundInflammatory bowel disease (IBD) pathogenesis is still not well understood. It is considered to result from genetic susceptibility, environment, microbiota composition and aberrant immune response. Crohn's disease (CD) and ulcerative colitis (UC), forms of IBD, are sometimes indistinguishable by typical laboratory and clinical characteristics making timely diagnosis and subsequent therapy hit-and-miss. Glycosylation has shown a promising biomarker potential for early IBD diagnosis and effective response to treatment prediction.Scope of reviewThis mini-review briefly covers present knowledge of IBD pathophysiology, with a focus on recent research on the role of glycosylation in IBD pathogenesis and disease progression.Major conclusionsAberrant glycosylation significantly changes functionality of key proteins in intestinal niche and is involved in IBD etiology.General significanceElucidating mechanisms of IBD development is one of critical goals in managing this disease. Glycans are important for fine-tuning of intestinal processes that ensure homeostatic conditions which, if disrupted, lead to IBD.     

Genetic defects in the hexosamine and sialic acid biosynthesis pathway

BackgroundCongenital disorders of glycosylation are caused by defects in the glycosylation of proteins and lipids. Classically, gene defects with multisystem disease have been identified in the ubiquitously expressed glycosyltransferases required for protein N-glycosylation. An increasing number of defects are being described in sugar supply pathways for protein glycosylation with tissue-restricted clinical symptoms.Scope of reviewIn this review, we address the hexosamine and sialic acid biosynthesis pathways in sugar metabolism. GFPT1, PGM3 and GNE are essential for synthesis of nucleotide sugars uridine diphosphate N-acetylglucosamine (UDP-GlcNAc) and cytidine-5′-monophospho-N-acetylneuraminic acid (CMP-sialic acid) as precursors for various glycosylation pathways. Defects in these enzymes result in contrasting clinical phenotypes of congenital myasthenia, immunodeficiency or adult-onset myopathy, respectively. We therefore discuss the biochemical mechanisms of known genetic defects in the hexosamine and CMP-sialic acid synthesis pathway in relation to the clinical phenotypes.Major conclusionsBoth UDP-GlcNAc and CMP-sialic acid are important precursors for diverse protein glycosylation reactions and for conversion into other nucleotide-sugars. Defects in the synthesis of these nucleotide sugars might affect a wide range of protein glycosylation reactions. Involvement of multiple glycosylation pathways might contribute to disease phenotype, but the currently available biochemical information on sugar metabolism is insufficient to understand why defects in these pathways present with tissue-specific phenotypes.General significanceFuture research on the interplay between sugar metabolism and different glycosylation pathways in a tissue- and cell-specific manner will contribute to elucidation of disease mechanisms and will create new opportunities for therapeutic intervention. This article is part of a Special Issue entitled "Glycans in personalised medicine" Guest Editor: Professor Gordan Lauc.     

综述: 卵巢癌生物标志物的糖链谱研究进展

糖类抗原125(CA125)被认为是卵巢癌诊断的“金标准”,但在临床应用中普遍存在着特异性不高的问题．肿瘤形成和发展过程中常伴有糖基化修饰异常和糖链结构的改变,不同的肿瘤具有特异的异常糖链结构．近年来,借助凝集素芯片、多重质谱分析等糖蛋白组学和糖组学研究技术,发现不同来源CA125的O-糖链和N-糖链结构存在着明显的微观不均一性,以这些特征性糖链结构为标志物,可以显著提高CA125对卵巢癌的诊断特异性．在过去的10年,研究者们除对CA125糖链结构和糖基化模式做了深入的研究外,还利用糖组的研究方法,直接对来自卵巢癌患者血液、体液(腹水、囊泡液等)中糖蛋白的糖链做了精细的结构解析,结果显示,可有效鉴别卵巢癌患者和健康志愿者的特异性N-糖链结构,有可能成为灵敏度高和特异性好的卵巢癌生物标志物．卵巢癌生物标志物研究发展的总趋势是从传统的对蛋白质的定性和定量研究,逐步转向于对标志物糖基化修饰和特异性糖链结构的鉴定以及定量分析．本文从糖组学的视角,对卵巢癌标志物糖组学的研究现状及发展趋势进行了综述和展望．