Glycans, often referred to as carbohydrates or sugars, are essential biomolecules found in all living organisms. Their structural diversity and complexity contribute to a wide range of biological functions, including cell-cell communication, immune response modulation, and pathogen recognition. Understanding the structure of glycans is crucial for unraveling their biological roles and developing therapeutic interventions targeting glycan-related diseases. In this comprehensive review, we explore the latest advancements in glycan structure determination techniques, highlighting their methodologies, applications, and future prospects.
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Molecular Weight Determination
Accurate determination of the molecular weight of glycans is essential for understanding their size, heterogeneity, and structural complexity. Mass spectrometry (MS) has emerged as the cornerstone technique for precisely determining the molecular weight of glycans. Traditional ionization methods, such as electron impact (EI) and chemical ionization (CI), often fail to adequately ionize sugars due to their low volatility and thermal instability. However, modern ionization techniques, including electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI), have revolutionized glycan analysis by enabling the generation of intact glycan ions ([M+H]+, [M+Na]+) suitable for precise mass determination.
Electrospray ionization (ESI) is particularly well-suited for glycan analysis due to its ability to generate multiply charged ions from polar and non-volatile analytes. In ESI, the sample solution is infused into the ion source under high voltage, resulting in the formation of a fine aerosol of charged droplets. As solvent evaporates from the droplets, analyte molecules become ionized and are transferred into the gas phase. The resulting ions are then analyzed by mass spectrometry, providing accurate mass measurements of glycan ions.
MALDI is another powerful ionization technique commonly used for glycan analysis. In MALDI, glycan samples are mixed with a matrix compound and deposited on a solid target. Upon irradiation with a laser beam, the matrix absorbs energy and transfers it to the analyte molecules, causing desorption and ionization. The resulting ions are then accelerated into the mass analyzer for mass measurement. MALDI is well-suited for high-throughput glycan analysis and is particularly useful for analyzing large, labile glycans.
In addition to accurate mass determination, tandem mass spectrometry (MS/MS) techniques play a crucial role in elucidating the structure of glycans. By subjecting glycan ions to fragmentation in the mass spectrometer, MS/MS provides valuable structural information about glycan residues and their linkage patterns. Fragmentation techniques such as collision-induced dissociation (CID) and electron transfer dissociation (ETD) break glycan ions into smaller fragments, allowing for the identification of glycan subunits and their connectivity.
Monosaccharide Identification
The identification and quantification of monosaccharide residues within glycan structures are essential for understanding their composition, diversity, and biological functions. Several methods are commonly employed for monosaccharide identification, each offering unique advantages and insights into glycan composition.
Acid hydrolysis followed by chromatographic separation is a classical approach for monosaccharide identification. In this method, glycan samples are subjected to acidic conditions, typically using hydrochloric acid or trifluoroacetic acid, to cleave glycosidic linkages and release monosaccharide units. The resulting mixture of monosaccharides is then separated and detected using chromatographic techniques, such as high-performance liquid chromatography (HPLC) or capillary electrophoresis (CE). HPLC separation offers excellent resolution and sensitivity, while CE provides rapid analysis with minimal sample consumption. Detection of monosaccharides is typically achieved using a variety of detectors, including refractive index (RI), ultraviolet (UV), and fluorescence detectors, depending on the specific analytical requirements.
An alternative approach for monosaccharide identification is derivatization followed by gas chromatography (GC) analysis. In this method, glycan samples are derivatized, typically through permethylation or trimethylsilylation, to convert hydroxyl groups into volatile derivatives amenable to GC analysis. Permethylation involves the methylation of hydroxyl groups using methyl iodide, while trimethylsilylation replaces hydroxyl groups with trimethylsilyl (TMS) groups. The derivatized monosaccharides are then analyzed using GC coupled with flame ionization detection (FID) or mass spectrometry (MS), allowing for the sensitive and selective detection of monosaccharides. GC analysis offers excellent resolution and specificity, making it particularly useful for complex glycan mixtures and trace-level analysis.
In addition to chromatographic methods, mass spectrometry (MS) techniques are increasingly being used for monosaccharide identification. Direct infusion MS and tandem MS (MS/MS) enable the rapid and sensitive detection of monosaccharides based on their mass-to-charge ratio (m/z) and fragmentation patterns. By comparing experimental spectra to reference databases or standards, monosaccharide residues within glycan structures can be confidently identified and quantified.
Overall, monosaccharide identification is a critical step in glycan analysis, providing valuable information about glycan composition and structure. The combination of chromatographic and mass spectrometry techniques offers complementary advantages for monosaccharide analysis, allowing researchers to comprehensively characterize the monosaccharide content of complex glycan samples with high sensitivity and specificity.
Determination of Glycosidic Linkage Positions
Accurate determination of glycosidic linkage positions is essential for elucidating the three-dimensional structure and functional properties of glycans. Several methodologies are commonly employed for determining glycosidic linkage positions, each offering unique advantages and insights into glycan structure.
One approach for determining glycosidic linkage positions involves chemical methods, such as partial acid hydrolysis and enzymatic cleavage. In partial acid hydrolysis, glycan samples are subjected to mild acidic conditions, typically using hydrochloric acid or trifluoroacetic acid, to selectively cleave glycosidic linkages at specific positions. The resulting mixture of oligosaccharides is then analyzed using chromatographic techniques, such as high-performance liquid chromatography (HPLC) or capillary electrophoresis (CE), to identify the position of cleavage. By comparing the retention times or electrophoretic mobilities of oligosaccharides to reference standards, glycosidic linkage positions can be determined with high accuracy.
Structural basis of diverse glycosidic linkages (Zhou et al., 2011).
Enzymatic cleavage is another powerful approach for determining glycosidic linkage positions. Glycan samples are incubated with specific glycosidases, enzymes that catalyze the hydrolysis of glycosidic linkages at specific positions, resulting in the release of monosaccharide or oligosaccharide fragments. The identity and position of glycosidic linkages are determined by analyzing the composition and sequence of the released fragments using chromatographic or mass spectrometry techniques. Enzymatic cleavage offers high specificity and selectivity, allowing for the precise determination of glycosidic linkage positions within complex glycan structures.
Nuclear magnetic resonance (NMR) spectroscopy is a powerful technique for elucidating glycosidic linkage positions at the atomic level. In NMR spectroscopy, glycan samples are dissolved in a suitable solvent and subjected to radiofrequency irradiation in the presence of a strong magnetic field. By measuring the chemical shifts and coupling constants of hydrogen and carbon atoms in the glycan structure, glycosidic linkage positions can be determined with high precision. Two-dimensional NMR techniques, such as correlation spectroscopy (COSY) and nuclear Overhauser effect spectroscopy (NOESY), enable the identification of spatial correlations between atoms, further facilitating the determination of glycosidic linkage positions within complex glycan structures.
In addition to experimental techniques, computational modeling and molecular dynamics simulations play a crucial role in predicting glycosidic linkage positions and elucidating glycan structure-function relationships. By combining experimental data with computational methods, researchers can gain deeper insights into the dynamic behavior and functional properties of glycans, ultimately leading to a better understanding of their biological roles and therapeutic potential.
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Elucidation of Glycan Connectivity Order
Determining the connectivity order of glycans is essential for understanding their biological functions, molecular recognition events, and structural properties. Several methodologies are commonly employed for elucidating glycan connectivity order, each offering unique advantages and insights into glycan structure.
Chemical degradation methods, such as Smith degradation and glycosidase digestion, are classical approaches for determining glycan connectivity order. In Smith degradation, glycan samples are subjected to controlled hydrolysis under mild acidic conditions, resulting in the stepwise cleavage of glycosidic linkages. By analyzing the composition and sequence of the resulting oligosaccharide fragments using chromatographic techniques, such as high-performance liquid chromatography (HPLC) or capillary electrophoresis (CE), the connectivity order of glycans can be deduced. Similarly, glycosidase digestion involves the enzymatic hydrolysis of glycan linkages by specific glycosidases, resulting in the release of monosaccharide or oligosaccharide fragments. The identity and sequence of these fragments are then determined using chromatographic or mass spectrometry techniques, allowing for the elucidation of glycan connectivity order.
Mass spectrometry (MS)-based techniques, such as collision-induced dissociation (CID) and electron transfer dissociation (ETD), are powerful tools for elucidating glycan connectivity order at the molecular level. By subjecting glycan ions to fragmentation in the mass spectrometer, MS/MS provides valuable structural information about glycan residues and their linkage patterns. Fragmentation techniques such as CID and ETD break glycan ions into smaller fragments, allowing for the identification of glycan subunits and their connectivity. By analyzing the fragmentation patterns and sequence of fragment ions, researchers can deduce the connectivity order of glycans with high accuracy.
Two-dimensional nuclear magnetic resonance (2D-NMR) spectroscopy and nuclear Overhauser effect (NOE) difference spectroscopy are advanced techniques for elucidating glycan connectivity order. In 2D-NMR spectroscopy, glycan samples are subjected to radiofrequency irradiation in the presence of a strong magnetic field, resulting in the generation of multidimensional NMR spectra. By analyzing the spatial correlations between atoms in the glycan structure, researchers can deduce the connectivity order of glycans with high precision. Similarly, NOE difference spectroscopy exploits the nuclear Overhauser effect to detect spatial correlations between atoms in the glycan structure, allowing for the determination of glycan connectivity order.
Determination of Glycosidic Bond Configuration:
Accurately determining the configuration of glycosidic bonds is pivotal for understanding the structural intricacies and functional implications of glycans. Several methodologies are employed for glycosidic bond configuration determination, each offering unique insights into glycan structure.
- Enzymatic Hydrolysis Method: Enzymatic hydrolysis is a widely-used method for determining glycosidic bond configuration. Specific enzymes, such as maltase and amygdalase, selectively cleave α- and β-glycosidic bonds, respectively. However, it's crucial to note that not all β-glycosidic bonds are hydrolyzed by amygdalase.
- Klyne Method: The Klyne method, also known as molecular rotation difference method, calculates glycosidic bond configuration based on the difference in specific rotation values between glycosides and their hydrolyzed glycosyl units. This empirical approach involves measuring the specific rotation of unknown glycosides and their corresponding glycosyl units, subtracting the specific rotation of glycosyl units from glycosides to obtain Δ[α], and comparing Δ[α] values with reference values for α- and β-glycosides.
- NMR Spectroscopy: Nuclear magnetic resonance (NMR) spectroscopy is utilized for glycosidic bond configuration determination based on proton-proton coupling constants (J-values) and carbon-proton coupling constants. In the 1H-NMR spectrum of sugars, the proton signals of terminal groups are typically observed around δ5.0, while those of sugar ring protons are between δ3.5-4.5. The J-values between C1-H and C2-H protons are indicative of glycosidic bond configuration. For most pyranose sugars like glucose, a J-value of 6-8 Hz suggests a β-glycosidic bond, while a J-value of 3-4 Hz indicates an α-glycosidic bond. However, for sugars like mannose and galactose, different configurations may have similar J-values, necessitating additional techniques such as selective irradiation or carbon-proton coupling constants for differentiation.
These methods, combined with complementary techniques like IR spectroscopy and mass spectrometry, provide comprehensive tools for determining the configuration of glycosidic bonds in complex glycan structures. Advancements in analytical instrumentation and computational modeling continue to enhance our ability to elucidate glycan structures with unprecedented accuracy and detail.
Reference
- Zhou, Dapeng, et al. "Immunologic mapping of glycomes: implications for cancer diagnosis and therapy." Frontiers in bioscience (Scholar edition) 3 (2011): 1520.