Glycomics involves investigating the isolation and purification of various glycans, including glycoproteins, proteoglycans, and glycolipids. This field covers the isolation of glycans specifically from glycoproteins, proteoglycans, and glycolipids. Additionally, it encompasses efforts in structural elucidation and quantification of these glycans. Furthermore, glycomics delves into exploring the properties and functions associated with glycans.
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Separation and Purification of Glycans
Separation of Glycoproteins
The separation of glycoproteins within the glycan category involves a two-step process. Initially, total proteins are isolated from biological samples, such as tissues or blood. Subsequently, glycoproteins are separated from the extracted total proteins for further investigation. Glycoproteins exhibit properties of both carbohydrates and proteins, and they are typically soluble in water, dilute acids, or dilute alkali solutions. Depending on specific requirements, various solvents can be employed for separation.
One common method for distinguishing glycoproteins from non-glycoproteins leverages the distinctive properties of glycoproteins. For instance, free sugars, monosaccharides, and oligosaccharides can selectively bind to lectins, and affinity chromatography can be utilized for separation. However, this method is limited to glycoproteins with specific glycan structures. Another approach involves employing two-dimensional gel electrophoresis in conjunction with fluorescence labeling techniques, which convert sugars into aldehydes for straightforward fluorescent labeling. While this method is more versatile and can target a broad range of glycan structures, it may alter glycan structures and is not suitable for glycan composition analysis.
When proteins are exposed to organic solvents like chloroform, they become denatured and insoluble in water, whereas sugars can be adsorbed after binding with dyes. Therefore, techniques such as the Sevage method, trichloroacetic acid method, trifluoroacetic acid method, and desalting can be employed to separate glycoproteins from non-glycoproteins.
Separation of Proteoglycans
Proteoglycans, a distinct class of glycoproteins, possess glycosaminoglycan (GAG) chains alongside O-linked or N-linked oligosaccharide chains. Extracellular matrix proteoglycans can be classified into high-molecular-weight and low-molecular-weight types based on the number of glycosaminoglycan chains they harbor. The intricate and diverse molecular structures and interactions of proteoglycans present a challenge in establishing a universal separation method, contributing to the limited progress in proteoglycan research.
Gel electrophoresis, owing to the high glycosaminoglycan content, strong hydrophilicity, and microheterogeneity of proteoglycan molecules, is not an ideal separation technique. Therefore, methods employing matrices with diverse pore sizes, such as polyacrylamide gel filtration (e.g., S-500 or S-1000), agarose gel filtration (e.g., CL-2B), or Superose-6, are utilized for the separation of different proteoglycan types. Additionally, lectin affinity chromatography proves useful in separating membrane-associated proteoglycans containing glycosaminoglycans.
Separation of Glycolipids
Among the diverse types of glycolipids, glycosphingolipids, incorporating sphingosine or glycerol lipids, hold a pivotal role in human physiology, biology, and medicine. To extract glycosphingolipids containing sphingosine or glycerol lipids from biological samples like tissues and bodily fluids, a standard two-step approach is often employed. Initially, total lipids are extracted from the biological samples, followed by the extraction of glycosphingolipids from the total lipids. This method capitalizes on the unique properties of glycosphingolipids, encompassing polar hydroxyl and carboxyl groups, as well as hydrophobic fatty acid chains.
Various methods can be utilized to isolate glycosphingolipids, including large-pore adsorbent resin column chromatography, column chromatography, thin-layer chromatography, high-performance liquid chromatography (HPLC), and reverse-phase chromatography. For the purification of specific types of glycosphingolipids from lipid extracts, techniques such as DEAE-Sephadex A-25, silica gel column chromatography, thin-layer chromatography, liquid-liquid partition chromatography, and liquid droplet counter-current chromatography can be implemented.
Structure Analysis of Glycans
The elucidation of glycan structures relies on various techniques such as mass spectrometry, nuclear magnetic resonance (NMR), chromatography, and lectin microarrays.
Mass Spectrometry
Mass spectrometry plays a crucial role in glycomics research. Commonly used mass spectrometry techniques include electrospray ionization mass spectrometry (ESI-MS), matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS), and Fourier-transform ion cyclotron resonance mass spectrometry (FT-ICR-MS).
Two primary derivatization methods are widely employed: full methylation and reductive amination. Full methylation involves reagents like iodomethane to convert the highly polar OH-, NH-, and COOH-groups on glycan chains into nonpolar OCH3-, NCH3-, and COOCH3-groups, enhancing the sensitivity of mass spectrometry. This method also enables the simultaneous detection of neutral and acidic glycans in positive ion mode MALDI-TOF-MS. A recent development is solid-phase full methylation with sodium hydroxide in capillaries or purification columns, which improves the reproducibility of derivatization analysis and has been successfully applied for quantitative analysis of glycan changes in different cancers, such as esophageal and ovarian cancer.
In reductive amination, derivatization reagents with active primary amino or hydrazide groups react with the reducing end aldehyde of glycan chains under acid-catalyzed conditions, forming Schiff bases. These Schiff bases are then reduced to stable single bonds using sodium borohydride (NaBH3CN) or similar reducing agents. Common reagents for reductive amination include 2-aminopyridine, aminobenzamide, ethyl anthranilate, and 1-aminopyrene-3,6,8-trisulfonate. Derivatization enhances the hydrophobicity of glycan chains, eliminating differences in ionization efficiency between neutral and acidic glycans and promoting the separation efficiency of mass spectrometry.
Multiple-stage MS/MS analysis of a hemagglutinin glycopeptide of the doubly charged ion of m/z 1309.5519 by LTQ-FT mass spectrometry (She et al., 2017).
Lectin Microarray Technology
Lectin microarray technology, based on the principle of microarrays, has been developed for glycomics research. This high-throughput method provides information about glycan structures. Among microarray technologies, lectin microarrays have seen rapid development. Typically, various plant lectins are immobilized on a solid support with defined spatial distances. Fluorescently labeled samples react with the lectins on the microarray, and labeled samples are specifically recognized through their own glycan chains, enabling analysis of the glycan structures by scanning the intensity of fluorescence.
Quantitative Analysis of Glycans
Quantitative studies of glycans encompass both absolute and relative quantification, frequently conducted alongside structural analysis. Absolute quantification involves determining the total content of a specific sugar or all sugars in one or more glycan samples. This method typically relies on the availability of standards. However, due to the intricate nature of glycan compositions in biological systems, achieving absolute quantification of glycans remains challenging, and no widely accepted methods exist.
In contrast, relative quantification does not entail determining the absolute content of sugars in glycan samples. Instead, it focuses on the relative content, comparing the quantities of a specific sugar or all sugars in two or more samples. Internal standard methods, including isotope labeling and metabolic labeling, are commonly employed for relative quantification.
Stable Isotope Labeling with Methyl Groups
Stable isotope labeling with methyl groups, coupled with mass spectrometry, is employed for the relative quantification analysis of glycans. This methodology involves chemically modifying glycans through derivatization and subsequent labeling with stable isotopes. Distinct samples containing glycans undergo labeling, after which they are combined in predetermined proportions and subjected to mass spectrometry analysis. Consequently, identical glycans with different isotope masses generate distinctive peaks. Relative quantification is accomplished by comparing the relative abundances of identical glycans from different sources.
This approach offers compatibility with various mass spectrometry techniques. However, it faces a limitation in necessitating uniform methylation efficiency. Achieving consistent methylation efficiency poses a challenge due to the presence of numerous hydroxyl, carboxyl, and amino groups in oligosaccharides. Ensuring accurate quantification requires meticulous attention to achieving identical methylation efficiency, which can be challenging.
Metabolic Labeling Method
In the metabolic labeling method, the nitrogen on the side chain of glutamine in hexosamine synthesis serves as the sole nitrogen source in the biosynthesis of N-acetylglucosamine, N-acetylgalactosamine, and sialic acid. By introducing heavy nitrogen (15N) labeled glutamine (15N-Gln) into the culture medium, 15N can be incorporated into all amino sugars in the cell during the metabolic process. This results in an increase of 1 atomic mass unit (U) for each nitrogen and oxygen-linked glycan, glycolipid, and amino sugar in extracellular matrix proteoglycans.
Quantification is achieved by comparing the signal intensities of peaks in glycan mass spectra from cells cultured in media with light and heavy glutamine. This method is known as the isotopic detection of glutamine-labeled amino sugars. It allows for relative quantification of glycan abundances.
Reference
- She, Yi-Min, et al. "Topological N-glycosylation and site-specific N-glycan sulfation of influenza proteins in the highly expressed H1N1 candidate vaccines." Scientific reports 7.1 (2017): 10232.
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