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.     

GlycReSoft: A Software Package for Automated Recognition of Glycans from LC/MS Data

Glycosylation modifies the physicochemical properties and protein binding functions of glycoconjugates. These modifications are biosynthesized in the endoplasmic reticulum and Golgi apparatus by a series of enzymatic transformations that are under complex control. As a result, mature glycans on a given site are heterogeneous mixtures of glycoforms. This gives rise to a spectrum of adhesive properties that strongly influences interactions with binding partners and resultant biological effects. In order to understand the roles glycosylation plays in normal and disease processes, efficient structural analysis tools are necessary. In the field of glycomics, liquid chromatography/mass spectrometry (LC/MS) is used to profile the glycans present in a given sample. This technology enables comparison of glycan compositions and abundances among different biological samples, i.e. normal versus disease, normal versus mutant, etc. Manual analysis of the glycan profiling LC/MS data is extremely time-consuming and efficient software tools are needed to eliminate this bottleneck. In this work, we have developed a tool to computationally model LC/MS data to enable efficient profiling of glycans. Using LC/MS data deconvoluted by Decon2LS/DeconTools, we built a list of unique neutral masses corresponding to candidate glycan compositions summarized over their various charge states, adducts and range of elution times. Our work aims to provide confident identification of true compounds in complex data sets that are not amenable to manual interpretation. This capability is an essential part of glycomics work flows. We demonstrate this tool, GlycReSoft, using an LC/MS dataset on tissue derived heparan sulfate oligosaccharides. The software, code and a test data set are publically archived under an open source license.     

Bioinformatics for glycomics: status, methods, requirements and perspectives

The term 'glycomics' describes the scientific attempt to identify and study all the glycan molecules - the glycome - synthesised by an organism. The aim is to create a cell-by-cell catalogue of glycosyltransferase expression and detected glycan structures. The current status of databases and bioinformatics tools, which are still in their infancy, is reviewed. The structures of glycans as secondary gene products cannot be easily predicted from the DNA sequence. Glycan sequences cannot be described by a simple linear one-letter code as each pair of monosaccharides can be linked in several ways and branched structures can be formed. Few of the bioinformatics algorithms developed for genomics/proteomics can be directly adapted for glycomics. The development of algorithms, which allow a rapid, automatic interpretation of mass spectra to identify glycan structures is currently the most active field of research. The lack of generally accepted ways to normalise glycan structures and exchange glycan formats hampers an efficient cross-linking and the automatic exchange of distributed data. The upcoming glycomics should accept that unrestricted dissemination of scientific data accelerates scientific findings and initiates a number of new initiatives to explore the data.     

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.     

Versatile fluorescent derivatization of glycans for glycomic analysis

The new field of functional glycomics encompasses information about both glycan structure and recognition by carbohydrate-binding proteins (CBPs) and is now being explored through glycan array technology. Glycan array construction, however, is limited by the complexity of efficiently generating derivatives of free, reducing glycans with primary amines for conjugation. Here we describe a straightforward method to derivatize glycans with 2,6-diaminopyridine (DAP) to generate fluorescently labeled glycans (glycan-DAP conjugates or GDAPs) that contain a primary amine for further conjugation. We converted a wide variety of glycans, including milk sugars, N-glycans, glycosaminoglycans and chitin-derived glycans, to GDAPs, as verified by HPLC and mass spectrometry. We covalently conjugated GDAPs to N-hydroxysuccinimide (NHS)-activated glass slides, maleimide-activated protein, carboxylated microspheres and NHS-biotin to provide quantifiable fluorescent derivatives. All types of conjugated glycans were well-recognized by appropriate CBPs. Thus, GDAP derivatives provide versatile new tools for biologists to quantify and covalently capture minute quantities of glycans for exploring their structures and functions and generating new glycan arrays from naturally occurring glycans.     

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."     

Glycomics: an integrated systems approach to structure-function relationships of glycans

In comparison with genomics and proteomics, the advancement of glycomics has faced unique challenges in the pursuit of developing analytical and biochemical tools and biological readouts to investigate glycan structure-function relationships. Glycans are more diverse in terms of chemical structure and information density than are DNA and proteins. This diversity arises from glycans' complex nontemplate-based biosynthesis, which involves several enzymes and isoforms of these enzymes. Consequently, glycans are expressed as an 'ensemble' of structures that mediate function. Moreover, unlike protein-protein interactions, which can be generally viewed as 'digital' in regulating function, glycan-protein interactions impinge on biological functions in a more 'analog' fashion that can in turn 'fine-tune' a biological response. This fine-tuning by glycans is achieved through the graded affinity, avidity and multivalency of their interactions. Given the importance of glycomics, this review focuses on areas of technologies and the importance of developing a bioinformatics platform to integrate the diverse datasets generated using the different technologies to allow a systems approach to glycan structure-function relationships.     

Integrated mass spectrometric strategy for characterizing the glycans from glycosphingolipids and glycoproteins: direct identification of sialyl Le(x) in mice

The current interest in applying systems biology approaches to studying an organism's form or function promises to reveal further insights into the role of glycosylation in cells and whole organisms. This has prompted the development of a rapid, sensitive method of profiling the glycan component of both glycosphingolipids and glycoproteins from a single sample. Here we report a new mass spectrometric screening strategy for characterizing glycosphingolipid-derived oligosaccharides, which can be integrated into an existing highly sensitive glycoprotein glycomics strategy. Using ceramide glycanase to release the glycans from glycosphingolipids, this method provides a reliable profile of the glycosphingolipid-derived glycans present in a sample and has revealed new glycan structures. Glycoproteins are also efficiently recovered using this method, allowing the subsequent analysis of glycoprotein-derived glycans by mass spectrometry. The high sensitivity of this glycomic screening method allowed us to directly characterize the sialyl Le(x) epitope from mouse brain for the first time, where it was observed on an O-mannose structure. Thus, we present a mass spectrometric method that allows glycomic screening of N- and O-glycans as well as glycosphingolipid-derived glycans from a single tissue.     

A versatile method for functionalizing surfaces with bioactive glycans

Microarrays and biosensors owe their functionality to our ability to display surface-bound biomolecules with retained biological function. Versatile, stable, and facile methods for the immobilization of bioactive compounds on surfaces have expanded the application of high-throughput "omics"-scale screening of molecular interactions by nonexpert laboratories. Herein, we demonstrate the potential of simplified chemistries to fabricate a glycan microarray, utilizing divinyl sulfone (DVS)-modified surfaces for the covalent immobilization of natural and chemically derived carbohydrates, as well as glycoproteins. The bioactivity of the captured glycans was quantitatively examined by surface plasmon resonance imaging (SPRi). Composition and spectroscopic evidence of carbohydrate species on the DVS-modified surface were obtained by X-ray photoelectron spectroscopy (XPS) and time-of-flight secondary ion mass spectrometry (ToF-SIMS), respectively. The site-selective immobilization of glycans based on relative nucleophilicity (reducing sugar vs amine- and sulfhydryl-derived saccharides) and anomeric configuration was also examined. Our results demonstrate straightforward and reproducible conjugation of a variety of functional biomolecules onto a vinyl sulfone-modified biosensor surface. The simplicity of this method will have a significant impact on glycomics research, as it expands the ability of nonsynthetic laboratories to rapidly construct functional glycan microarrays and quantitative biosensors.     

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.     

Identification of unique glycoisoforms of vitamin D-binding protein and haptoglobin as biomarker candidates in hepatocarcinogenesis of STAM mice

Hepatocellular carcinoma (HCC) is the major subtype of primary liver cancer, and is typically diagnosed late in its course. Considering the limitations and the reluctance of patients to undergo a liver biopsy, a reliable, noninvasive diagnostic marker that predicts and assesses the treatment and prognosis of HCC is needed. With recent technological advances of mass spectrometry, glycomics is gathering momentum and holds substantial potential to discover new glycan markers in cancer research. Here, to discover specific glycan markers for the early diagnosis of HCC, we analyzed the glycan profiles of gel-separated serum proteins of progressive liver disease model mice. By focused protein glycomics of 12 gel-separated glycoproteins using sera from the mouse models, we revealed the entire profile of glycans in each major serum protein. We found that the levels of trisialylated triantennary glycans of haptoglobin and vitamin D-binding protein increased significantly as the disease progressed, while the alteration in these protein levels were modest. Furthermore, these glycan increases were not observed in age-matched control mice. In conclusion, our approach has identified specific glycan marker candidates for the early diagnosis of HCC.     

Human disease glycomics: technology advances enabling protein glycosylation analysis – part 1

Introduction: Protein glycosylation is recognized as an important post-translational modification, with specific substructures having significant effects on protein folding, conformation, distribution, stability and activity. However, due to the structural complexity of glycans, elucidating glycan structure-function relationships is demanding. The fine detail of glycan structures attached to proteins (including sequence, branching, linkage and anomericity) is still best analysed after the glycans are released from the purified or mixture of glycoproteins (glycomics). The technologies currently available for glycomics are becoming streamlined and standardized and many features of protein glycosylation can now be determined using instruments available in most protein analytical laboratories.

Areas covered: This review focuses on the current glycomics technologies being commonly used for the analysis of the microheterogeneity of monosaccharide composition, sequence, branching and linkage of released N- and O-linked glycans that enable the determination of precise glycan structural determinants presented on secreted proteins and on the surface of all cells.

Expert commentary: Several emerging advances in these technologies enabling glycomics analysis are discussed. The technological and bioinformatics requirements to be able to accurately assign these precise glycan features at biological levels in a disease context are assessed.     

Strategy for fluorescent labeling of human acidic fibroblast growth factor without impairment of mitogenic activity: a bona fide tracer

A deuterium reagent, 1-(d5) phenyl-3-methyl-5-pyrazolone (d5-PMP), has been synthesized and used for relative quantitative analysis of oligosaccharides by mass spectrometry (MS) using d0/d5-PMP stable isotopic labeling. Previously reported permethylation-based isotopic labels generate variable mass differences, and reductive amination-based isotopic labels cause a loss of some acid-labile groups in carbohydrates. In contrast, d0/d5-PMP stable isotopic labeling is performed at the reducing end of glycans under basic conditions without desialylation, and the mass difference (Δm = 10 Da) between the heavy form (d5-PMP derivative) and light form (d0-PMP derivative) of each glycan is invariable. When the two derivative forms of a glycan are mixed in equimolar amounts, a pair of peaks with a 10-Da mass differences is observed in the MS profile. The difference at relative intensity between the d0- and d5-PMP derivatives reflects the difference in quantity of glycans in two samples, making it possible to carry out both qualitative and relative quantitative analyses of glycans in glycomic studies. Application of this method on DP₂ to DP₆ maltodextrin oligosaccharides and N-linked glycans released from ribonuclease B and bovine fetuin demonstrates a 10-fold relative quantitative dynamic range, a satisfying reproducibility (coefficient of variation [CV] ? 8.34%), and good accuracy (relative error [RE] ? 5.1%) of the method. The suggested technique has been successfully applied for comparative quantitative analysis of free oligosaccharides in human and bovine milk.     

From glycomics to functional glycomics of sugar chains: Identification of target proteins with functional changes using gene targeting mice and knock down cells of FUT8 as examples

Comprehensive analyses of proteins from cells and tissues are the most effective means of elucidating the expression patterns of individual disease-related proteins. On the other hand, the simultaneous separation and characterization of proteins by 1-DE or 2-DE followed by MS analysis are one of the fundamental approaches to proteomic analysis. However, these analyses do not permit the complete structural identification of glycans in glycoproteins or their structural characterization. Over half of all known proteins are glycosylated and glycan analyses of glycoproteins are requisite for fundamental proteomics studies. The analysis of glycan structural alterations in glycoproteins is becoming increasingly important in terms of biomarkers, quality control of glycoprotein drugs, and the development of new drugs. However, usual approach such as proteoglycomics, glycoproteomics and glycomics which characterizes and/or identifies sugar chains, provides some structural information, but it does not provide any information of functionality of sugar chains. Therefore, in order to elucidate the function of glycans, functional glycomics which identifies the target glycoproteins and characterizes functional roles of sugar chains represents a promising approach. In this review, we show examples of functional glycomics technique using alpha 1,6 fucosyltransferase gene (Fut8) in order to identify the target glycoprotein(s). This approach is based on glycan profiling by CE/MS and LC/MS followed by proteomic approaches, including 2-DE/1-DE and lectin blot techniques and identification of functional changes of sugar chains.     

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.     

Altered glycosylation in cancer: A promising target for biomarkers and therapeutics

Glycosylation is a well-regulated cell and microenvironment specific post-translational modification. Several glycosyltransferases and glycosidases orchestrate the addition of defined glycan structures on the proteins and lipids. Recent advances and systemic approaches in glycomics have significantly contributed to a better understanding of instrumental roles of glycans in health and diseases. Emerging research evidence recognized aberrantly glycosylated proteins as the modulators of the malignant phenotype of cancer cells. The Cancer Genome Atlas has identified alterations in the expressions of glycosylation-specific genes that are correlated with cancer progression. However, the mechanistic basis remains poorly explored. Recent researches have shown that specific changes in the glycan structures are associated with 'stemness' and epithelial-to-mesenchymal transition of cancer cells. Moreover, epigenetic changes in the glycosylation pattern make the tumor cells capable of escaping immunosurveillance mechanisms. The deciphering roles of glycans in cancer emphasize that glycans can serve as a source for the development of novel clinical biomarkers. The ability of glycans in intervening various stages of tumor progression and the biosynthetic pathways involved in glycan structures constitute a promising target for cancer therapy. Advances in the knowledge of innovative strategies for identifying the mechanisms of glycan-binding proteins are hoped to hold great potential in cancer therapy. This review discusses the fundamental role of glycans in regulating tumorigenesis and tumor progression and provides insights into the influence of glycans in the current tactics of targeted therapies in the clinical setting.     

Improvement of electrospray stability in negative ion mode for nano-PGC-LC-MS glycoanalysis via post-column make-up flow

Analysis of glycans via a porous graphitized carbon liquid chromatography (PGC-LC) coupled with electrospray ionization (tandem) mass spectrometry (ESI-MS(/MS)) is a powerful analytical method in the field of glycomics. Isobaric glycan structures can be identified reliably with the help of PGC-LC separation and subsequent identification by ESI-MS(/MS) in negative ion mode. In an effort to adapt PGC-LC-ESI-MS(/MS) to the nano-scale operation, spray instability along the nano-PGC-LC gradient was repeatedly observed on an LTQ Orbitrap Elite mass spectrometer equipped with a standard nano-electrospray ionization source. A stable electrospray was achieved with the implementation of a post-column make-up flow (PCMF). Thereby, acetonitrile was used to supplement the eluate from the nano-PGC-LC column. The improved spray stability enhanced detection and resolution of glycans during the analysis. This was in particular the case for smaller O-glycans which elute early in the high aqueous content regime of the nano-PGC-LC elution gradient. This study introduces PCMF as an easy-to-use instrumental adaptation to significantly improve spray stability in negative ion mode nano-PGC-LC-ESI-MS(/MS)-based analysis of glycans.     

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.     

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.     

Glycomics and Proteomics Approaches to Investigate Early Adenovirus–Host Cell Interactions

Adenoviruses as most viruses rely on glycan and protein interactions to attach to and enter susceptible host cells. The Adenoviridae family comprises more than 80 human types and they differ in their attachment factor and receptor usage, which likely contributes to the diverse tropism of the different types. In the past years, methods to systematically identify glycan and protein interactions have advanced. In particular sensitivity, speed and coverage of mass spectrometric analyses allow for high-throughput identification of glycans and peptides separated by liquid chromatography. Also, developments in glycan microarray technologies have led to targeted, high-throughput screening and identification of glycan-based receptors. The mapping of cell surface interactions of the diverse adenovirus types has implications for cell, tissue, and species tropism as well as drug development. Here we review known adenovirus interactions with glycan- and protein-based receptors, as well as glycomics and proteomics strategies to identify yet elusive virus receptors and attachment factors. We finally discuss challenges, bottlenecks, and future research directions in the field of non-enveloped virus entry into host cells.