Sweet spots in functional glycomics

Information contained in the mammalian glycome is decoded by glycan-binding proteins (GBPs) that mediate diverse functions including host-pathogen interactions, cell trafficking and transmembrane signaling. Although information on the biological roles of GBPs is rapidly expanding, challenges remain in identifying the glycan ligands and their impact on GBP function. Protein-glycan interactions are typically low affinity, requiring multivalent interactions to achieve a biological effect. Though many glycoproteins can carry the glycan structure recognized by the GBP, other factors, such as recognition of protein epitopes and microdomain localization, may restrict which glycoproteins are functional ligands in situ. Recent advances in development of glycan arrays, synthesis of multivalent glycan ligands, bioengineering of cell-surface glycans and glycomics databases are providing new tools to identify the ligands of GBPs and to elucidate the mechanisms by which they participate in GBP function.     

Shotgun glycomics: a microarray strategy for functional glycomics

Major challenges of glycomics are to characterize a glycome and identify functional glycans as ligands for glycan-binding proteins (GBPs). To address these issues we developed a general strategy termed shotgun glycomics. We focus on glycosphingolipids (GSLs), a class of glycoconjugates that is challenging to study, recognized by toxins, antibodies and GBPs. We derivatized GSLs extracted from cells with a heterobifunctional fluorescent tag suitable for covalent immobilization. We separated fluorescent GSLs by multidimensional chromatography, quantified them and coupled them to glass slides to create GSL shotgun microarrays. Then we interrogated the microarrays with cholera toxin, antibodies and sera from individuals with Lyme disease to identify biologically relevant GSLs that we subsequently characterized by mass spectrometry. Shotgun glycomics incorporating GSLs and potentially glycoprotein-derived glycans is an approach for accessing the complex glycomes of animal cells and is a strategy for focusing structural analyses on functionally important glycans.     

Lectin-based structural glycomics: Glycoproteomics and glycan profiling

Structural glycomics (SG) plays a fundamental part of concurrent glycobiology aiming at comprehensive elucidation of glycan functions ( i.e. , functional glycomics) in the context of post-genome sciences. The SG project started in April 2003 and will continue for 3 years in the framework of NEDO (New Energy and Industrial Technology Organization) under the METI (the Ministry of Economy, Trade, and Industry), Japan. The main purpose of the project is the development of high-throughput and robust machines, which should greatly contribute to the structural analysis of complex glycans. In this chapter, 2 major research items, i.e. , (1) glycoproteomics, which enables comprehensive analysis of glycoproteins, and (2) "glycan profiling" by means of lectins, are described. For the latter, frontal affinity chromatography has been adopted as a starting tool for comprehensive analysis of the interaction of 100 lectins and 100 oligosaccharides under the concept of "hect-by-hect," which refers to 100 x 100.     

A serum glycomics approach to breast cancer biomarkers

Because the glycosylation of proteins is known to change in tumor cells during the development of breast cancer, a glycomics approach is used here to find relevant biomarkers of breast cancer. These glycosylation changes are known to correlate with increasing tumor burden and poor prognosis. Current antibody-based immunochemical tests for cancer biomarkers of ovarian (CA125), breast (CA27.29 or CA15-3), pancreatic, gastric, colonic, and carcinoma (CA19-9) target highly glycosylated mucin proteins. However, these tests lack the specificity and sensitivity for use in early detection. This glycomics approach to find glycan biomarkers of breast cancer involves chemically cleaving oligosaccharides (glycans) from glycosylated proteins that are shed or secreted by breast cancer tumor cell lines. The resulting free glycan species are analyzed by MALDI-FT-ICR MS. Further structural analysis of the glycans can be performed in FTMS through the use of tandem mass spectrometry with infrared multiphoton dissociation. Glycan profiles were generated for each cell line and compared. These methods were then used to analyze sera obtained from a mouse model of breast cancer and a small number of serum samples obtained from human patients diagnosed with breast cancer or patients with no known history of breast cancer. In addition to the glycosylation changes detected in mice as mouse mammary tumors developed, glycosylation profiles were found to be sufficiently different to distinguish patients with cancer from those without. Although the small number of patient samples analyzed so far is inadequate to make any legitimate claims at this time, these promising but very preliminary results suggest that glycan profiles may contain distinct glycan biomarkers that may correspond to glycan "signatures of cancer."     

On the analysis of glycomics mass spectrometry data via the regularized area under the ROC curve

Background  

Novel molecular and statistical methods are in rising demand for disease diagnosis and prognosis with the help of recent advanced biotechnology. High-resolution mass spectrometry (MS) is one of those biotechnologies that are highly promising to improve health outcome. Previous literatures have identified some proteomics biomarkers that can distinguish healthy patients from cancer patients using MS data. In this paper, an MS study is demonstrated which uses glycomics to identify ovarian cancer. Glycomics is the study of glycans and glycoproteins. The glycans on the proteins may deviate between a cancer cell and a normal cell and may be visible in the blood. High-resolution MS has been applied to measure relative abundances of potential glycan biomarkers in human serum. Multiple potential glycan biomarkers are measured in MS spectra. With the objection of maximizing the empirical area under the ROC curve (AUC), an analysis method was considered which combines potential glycan biomarkers for the diagnosis of cancer.     

Glycan reductive isotope labeling for quantitative glycomics

Many diseases and disorders are characterized by quantitative and/or qualitative changes in complex carbohydrates. Mass spectrometry methods show promise in monitoring and detecting these important biological changes. Here we report a new glycomics method, termed glycan reductive isotope labeling (GRIL), where free glycans are derivatized by reductive amination with the differentially coded stable isotope tags [¹²C₆]aniline and [¹³C₆]aniline. These dual-labeled aniline-tagged glycans can be recovered by reverse-phase chromatography and can be quantified based on ultraviolet (UV) absorbance and relative ion abundances. Unlike previously reported isotopically coded reagents for glycans, GRIL does not contain deuterium, which can be chromatographically resolved. Our method shows no chromatographic resolution of differentially labeled glycans. Mixtures of differentially tagged glycans can be directly compared and quantified using mass spectrometric techniques. We demonstrate the use of GRIL to determine relative differences in glycan amount and composition. We analyze free glycans and glycans enzymatically or chemically released from a variety of standard glycoproteins, as well as human and mouse serum glycoproteins, using this method. This technique allows linear relative quantitation of glycans over a 10-fold concentration range and can accurately quantify sub-picomole levels of released glycans, providing a needed advancement in the field of glycomics.     

Advancing glycomics: implementation strategies at the consortium for functional glycomics

Glycomics-an integrated approach to study structure-function relationships of complex carbohydrates (or glycans)-is an emerging field in this age of post-genomics. Realizing the importance of glycomics, many large scale research initiatives have been established to generate novel resources and technologies to advance glycomics. These initiatives are generating and cataloging diverse data sets necessitating the development of bioinformatic platforms to acquire, integrate, and disseminate these data sets in a meaningful fashion. With the consortium for functional glycomics (CFG) as the model system, this review discusses databases and the bioinformatics platform developed by this consortium to advance glycomics.     

Investigation of cationic peanut peroxidase glycans by electrospray ionization mass spectrometry

Cationic peanut peroxidase (CP) was isolated from peanut (Arachis hypogaea) cell suspension culture medium. CP is a glycoprotein with three N-linked glycan sites at Asn60, Asn144, and Asn185. ESI-MS of the intact purified protein reveals the microheterogeneity of the glycans. Tryptic digestion of CP gave a near complete sequence coverage by ESI-MS. The glycopeptides from the tryptic digestion were separated by RP HPLC identified by ESI-MS and the structure of the glycan chains determined by ESI-MS/MS. The glycans are large structures of up to 16 sugars, but most of their non-reducing ends have been modified giving a mixture of shorter chains at each site. Good agreement was found with the one glycan previously analyzed by (1)H NMR. This work is the basis for the future studies on the role of the glycans on stability and folding of CP and is another example of a detailed structural characterization of complex glycoproteins by mass spectrometry.     

Emerging glycomics technologies

The nascent field of glycomics is currently undergoing rapid development, largely as a result of advances in technologies for analyzing glycan structure, unraveling glycan-protein interactions and establishing the functional significance of glycans. A meeting was held in November 2006 to explore the challenges and opportunities ahead for this emerging 'omics' domain.     

Quantitation by isobaric labeling: applications to glycomics

The study of glycosylation patterns (glycomics) in biological samples is an emerging field that can provide key insights into cell development and pathology. A current challenge in the field of glycomics is to determine how to quantify changes in glycan expression between different cells, tissues, or biological fluids. Here we describe a novel strategy, quantitation by isobaric labeling (QUIBL), to facilitate comparative glycomics. Permethylation of a glycan with (13)CH 3I or (12)CH 2DI generates a pair of isobaric derivatives, which have the same nominal mass. However, each methylation site introduces a mass difference of 0.002922 Da. As glycans have multiple methylation sites, the total mass difference for the isobaric pair allows separation and quantitation at a resolution of approximately 30000 m/Delta m. N-Linked oligosaccharides from a standard glycoprotein and human serum were used to demonstrate that QUIBL facilitates relative quantitation over a linear dynamic range of 2 orders of magnitude and permits the relative quantitation of isomeric glycans. We applied QUIBL to quantitate glycomic changes associated with the differentiation of murine embryonic stem cells to embryoid bodies.     

微阵列技术在糖组学研究中的应用

糖组学是研究糖链组成及其功能的一门新学科,近年来备受关注.目前糖组学的研究还处于起步阶段,阻碍糖组学迅速发展的主要原因是糖链本身结构的复杂性和研究技术的限制.微阵列技术作为一种快速、高效、高通量、微型化和自动化的分析技术,已经在基因组学和蛋白质组学的研究中发挥了重要的作用,将其应用于糖组学研究必将推动糖组学的发展.     

A two-dimensional array for simultaneous sequencing of N- and o-glycans and their glycoforms on specific glycosylation sites

Glycans play major roles in living organisms. Thus, essential information is required on diverse glycans, their location, and moieties in proteins, as well as for technology in a high-throughput manner, for improving functional glycomics. In the present study, we describe a new approach involving a 2-D array, which has the potential to fulfill both requirements. The first dimension of the array is composed of various lectins immobilized to a MALDI plate. The second dimension consists of initial proteolysis, then sequential exoglycosidase digestion using highly specific enzymes. The products of such digestions are peptide/glycopeptide mixtures conjugating different glycan fragments from which the exoglycosidase has removed specific terminal residues. Consequently, a series of spectra are obtained when lectin-attached products are analyzed by MALDI-TOF MS. By using well-known glycoproteins and NKp46D2-Ig, a recombinant fusion natural killer receptor with unknown glycans produced in CHO cells, we proved the usefulness of the method, demonstrating rapid and simultaneous determination of N- and O-glycan sequences, their glycan moieties, and subtypes on each of the determined glycosylation sites. This strategy provides a tool that can rapidly explore glycan structures and might contribute to a better understanding of process- and disease-related glycoproteins.     

Microwave-assisted nonspecific proteolytic digestion and controlled methylation for glycomics applications

Nonspecific proteolytic digestion of glycoproteins is an established technique in glycomics and glycoproteomics. In the presence of pronase E, for example, glycoproteins are digested to small glycopeptides having one to six amino acids residues, which can be analyzed with excellent sensitivity using mass spectrometry. Unfortunately, the long digestion times (1-3 days) limit the analytical throughput. In this study, we used controlled microwave irradiation to accelerate the proteolytic cleavage of glycoproteins mediated by pronase E. We used ESI-MS and MALDI-MS analyses to evaluate the microwave-assisted enzymatic digestions at various digestion durations, temperatures, and enzyme-to-protein ratios. When digesting glycoproteins, pronase E produced glycopeptides within 5 min under microwave irradiation; glycopeptides having one or two amino acids were the major products. Although analysis of peptides containing multiple amino acid residues offers the opportunity for peptide sequencing and provides information regarding the sites of glycosylation, the signals of Asn-linked glycans were often suppressed by the glycopeptides containing basic amino acids (Lys or Arg) in MALDI-MS experiments. To minimize this signal-to-content dependence, we converted the glycopeptides into their sodiated forms and then methylated them using methyl iodide. This controlled methylation procedure resulted in quaternization of the amino group of the N-terminal amino acid residue. Using this approach, the mass spectrometric response of glyco-Asn was enhanced, compensating for the poorer ionization efficiency associated with the basic amino acids residues. The methylated products of glycopeptides containing two or more amino acid residues were more stable than those containing only a single Asn residue. This feature can be used to elucidate glycan structures and glycosylation sites without the need for MS/MS analysis.     

Twoplex 12/13C6 aniline stable isotope and linkage‐specific sialic acid labeling 2D‐LC‐MS workflow for quantitative N‐glycomics

Quantitative glycomics represents an actively expanding research field ranging from the discovery of disease‐associated glycan alterations to the quantitative characterization of N‐glycans on therapeutic proteins. Commonly used analytical platforms for comparative relative quantitation of complex glycan samples include MALDI‐TOF‐MS or chromatographic glycan profiling with subsequent data alignment and statistical evaluation. Limitations of such approaches include run‐to‐run technical variation and the potential introduction of subjectivity during data processing. Here, we introduce an offline 2D LC‐MS^E workflow for the fractionation and relative quantitation of twoplex isotopically labeled N‐linked oligosaccharides using neutral ¹²C₆ and ¹³C₆ aniline (Δmass = 6 Da). Additional linkage‐specific derivatization of sialic acids using 4‐(4,6‐dimethoxy‐1,3,5‐trizain‐2‐yl)‐4‐methylmorpholinium chloride offered simultaneous and advanced in‐depth structural characterization. The potential of the method was demonstrated for the differential analysis of structurally defined N‐glycans released from serum proteins of patients diagnosed with various stages of colorectal cancer. The described twoplex ¹²C₆/¹³C₆ aniline 2D LC‐MS platform is ideally suited for differential glycomic analysis of structurally complex N‐glycan pools due to combination and analysis of samples in a single LC‐MS injection and the associated minimization in technical variation.     

Mass spectrometric profiling of O-linked glycans released directly from glycoproteins in gels using in-gel reductive beta-elimination

Glycosylation is a widespread PTM of proteins; the carbohydrate moieties provide various functional, immunological and structural aspects of both eukaryotic and prokaryotic glycoproteins. Traditional strategies used to analyse glycoprotein O-glycans involve glycoprotein isolation, followed by glycan release using solution-phase base-catalysed beta-elimination. However, in a proteomics context, mixtures of proteins and glycoproteins are routinely separated using SDS-PAGE. We have therefore developed a method to enable the profiling of O-linked glycans directly from glycoproteins on gels. This is achieved using in-gel reductive beta-elimination followed by mass spectrometric analysis of the released glycans. Here we describe our demonstration of the feasibility of this approach, our development and optimisation of the procedure using bovine submaxillary gland glycoproteins as a standard, and then show its usefulness by applying the developed procedure to the analysis of the O-glycans from a glycoprotein band from a Coomassie-stained SDS-PAGE separation of a mixture of Mycobacterium avium capsular proteins and glycoproteins. The procedure has been shown to be applicable to both CBB- and silver-stained gels. The method offers a quick and easy way to identify the O-glycans from gel-separated glycoproteins within gel-based proteomics workflows.     

Proteomics and glycomics analyses of N-glycosylated structures involved in Toxoplasma gondii--host cell interactions

The apicomplexan parasite Toxoplasma gondii recognizes, binds, and penetrates virtually any kind of mammalian cell using a repertoire of proteins released from late secretory organelles and a unique form of gliding motility (also named glideosome) that critically depends on actin filaments and myosin. How T. gondii glycosylated proteins mediate host-parasite interactions remains elusive. To date, only limited evidence is available concerning N-glycosylation in apicomplexans. Here we report comprehensive proteomics and glycomics analyses showing that several key components required for host cell-T. gondii interactions are N-glycosylated. Detailed structural characterization confirmed that N-glycans from T. gondii total protein extracts consist of oligomannosidic (Man(5-8)(GlcNAc)2) and paucimannosidic (Man(3-4)(GlcNAc)2) sugars, which are rarely present on mature eukaryotic glycoproteins. In situ fluorescence using concanavalin A and Pisum sativum agglutinin predominantly stained the entire parasite body. Visualization of Toxoplasma glycoproteins purified by affinity chromatography followed by detailed proteomics and glycan analyses identified components involved in gliding motility, moving junction, and other additional functions implicated in intracellular development. Importantly tunicamycin-treated parasites were considerably reduced in motility, host cell invasion, and growth. Collectively these results indicate that N-glycosylation probably participates in modifying key proteins that are essential for host cell invasion by T. gondii.     

S-layer nanoglycobiology of bacteria

Cell surface layers (S-layers) are common structures of the bacterial cell envelope with a lattice-like appearance that are formed by a self-assembly process. Frequently, the constituting S-layer proteins are modified with covalently linked glycan chains facing the extracellular environment. S-layer glycoproteins from organisms of the Bacillaceae family possess long, O-glycosidically linked glycans that are composed of a great variety of sugar constituents. The observed variations already exceed the display found in eukaryotic glycoproteins. Recent investigations of the S-layer protein glycosylation process at the molecular level, which has lagged behind the structural studies due to the lack of suitable molecular tools, indicated that the S-layer glycoprotein glycan biosynthesis pathway utilizes different modules of the well-known biosynthesis routes of lipopolysaccharide O-antigens. The genetic information for S-layer glycan biosynthesis is usually present in S-layer glycosylation (slg) gene clusters acting in concert with housekeeping genes. To account for the nanometer-scale cell surface display feature of bacterial S-layer glycosylation, we have coined the neologism 'nanoglycobiology'. It includes structural and biochemical aspects of S-layer glycans as well as molecular data on the machinery underlying the glycosylation event. A key aspect for the full potency of S-layer nanoglycobiology is the unique self-assembly feature of the S-layer protein matrix. Being aware that in many cases the glycan structures associated with a protein are the key to protein function, S-layer protein glycosylation will add a new and valuable component to an 'S-layer based molecular construction kit'. In our long-term research strategy, S-layer nanoglycobiology shall converge with other functional glycosylation systems to produce 'functional' S-layer neoglycoproteins for diverse applications in the fields of nanobiotechnology and vaccine technology. Recent advances in the field of S-layer nanoglycobiology have made our overall strategy a tangible aim of the near future.     

Mass spectrometry-based analysis of glycoproteins and its clinical applications in cancer biomarker discovery

Glycosylation is one of the most important posttranslational modifications of proteins and plays essential roles in various biological processes. Aberration in the glycan moieties of glycoproteins is associated with many diseases. It is especially critical to develop the rapid and sensitive methods for analysis of aberrant glycoproteins associated with diseases. Mass spectrometry (MS) has become a powerful tool for glycoprotein analysis. Especially, tandem mass spectrometry can provide highly informative fragments for structural identification of glycoproteins. This review provides an overview of the development of MS technologies and their applications in identification of abnormal glycoproteins and glycans in human serum to screen cancer biomarkers in recent years.     

Tools for glycomics: relative quantitation of glycans by isotopic permethylation using 13CH3I

Analysis of oligosaccharides by mass spectrometry (MS) has enabled the investigation of the glycan repertoire of organisms with high resolution and sensitivity. It is difficult, however, to correlate the expression of glycosyltransferases with the glycan structures present in a particular cell type or tissue because the use of MS for quantitative purposes has significant limitations. For this reason, in order to develop a technique that would allow relative glycan quantification by MS analysis between two samples, a procedure was developed for the isotopic labeling of oligosaccharides with (13)C-labeled methyl iodide using standard permethylation conditions. Separate aliquots of oligosaccharides from human milk were labeled with (12)C or (13)C methyl iodide; the labeled and non-labeled glycans were mixed in known proportions, and the mixtures analyzed by MS. Results indicated that the isotopic labeling described here was capable of providing relative quantitative data with a dynamic range of at least two orders of magnitude, adequate linearity, and reproducibility with a coefficient of variation that was 13% on average. This procedure was used to analyze N-linked glycans released from various mixtures of glycoproteins, such as alpha-1 acid glycoprotein, human transferrin, and bovine fetuin, using MS techniques that included matrix assisted laser desorption ionization-time of flight MS and electrospray ionization with ion cyclotron resonance-Fourier transformation MS. The measured (12)C:(13)C ratios from mixtures of glycans permethylated with either (12)CH(3)I or (13)CH(3)I were consistent with the theoretical proportions. This technique is an effective procedure for relative quantitative glycan analysis by MS.     

Structure, function and pathology of O-mannosyl glycans

Animal cells contain many glycoproteins, i.e. , proteins with covalently liked sugar chains. The major glycans of glycoproteins can be classified into two groups, N-glycans and O-glycans, according to their glycan-peptide linkage regions. Development of sensitive methods for the analyses of glycan structures have revealed a new type of glycosidic linkage to the peptide portion, the O-mannosyl linkage, in mammals, which used to be considered specific to yeast. O-Mannosylation is present in a limited number of glycoproteins of brain, nerve, and skeletal muscle. Recently O-mannosylation has been shown to be important in muscle and brain development. Glycobiology of O-mannosyl glycans is expected to produce remarkable advances in the understanding and treatment of congenital muscular dystrophies. In this article, I describe the structure, biosynthesis, and pathology of O-mannosyl glycans.