Glycan Derivatization Service

In the dynamic landscape of glycan research, the need for derivatization arises from the multifaceted nature of native glycan structures. These intricacies, encompassing isomeric variations and diverse structural forms, often hinder in-depth analysis using conventional methods. Our derivatization services act as a catalyst, simplifying these complexities and empowering your research with enhanced analytical sensitivity.

The Necessity for Glycan Derivatization

Enhanced Analytical Precision: Elevate your glycan analysis with derivatization, boosting sensitivity and precision in techniques like chromatography and mass spectrometry.

Structural Clarity: Derivatization serves as a powerful tool for modifying specific glycan functional groups, unraveling intricate structures, and decoding isomeric variations and branching patterns.

Efficient Chromatographic Separation: Modified glycans exhibit altered chromatographic behaviors, simplifying their separation in gas chromatography (GC) or liquid chromatography (LC) and enabling the resolution of complex glycan mixtures.

Improved Solubility and Stability: Increase glycan solubility and stability through derivatization, making them more compatible with diverse analytical techniques while minimizing the risk of degradation.

Precision Labeling for Detection: Derivatization introduces specific labels or tags for targeted glycan detection, allowing focused exploration of particular structural or functional aspects.

Quantitative Precision: Derivatization methods provide distinct and quantifiable signals, facilitating precise quantitative analysis and enabling the determination of glycan concentrations and ratios.

Techniques Employed in Glycan Derivatization

Fluorescent Labeling

Fluorescent labeling enhances glycan detectability by attaching fluorophores like 2-aminobenzamide and anthranilic acid. This technique aids sensitive detection in analytical methods, crucial for glycan quantification and visualization.

Permethylation

Permethylation replaces labile hydrogen atoms with methyl groups, crucial for mass spectrometry analysis. This technique stabilizes glycans, ensuring accurate mass spectra and supporting detailed structural analysis in glycomic studies.

Reducing-End Modification

Modifying the reducing end through reductive amination introduces functional groups, enhancing glycan versatility. This technique allows for selective detection and broadens downstream applications.

Linker-Based Derivatization

Linker-based derivatization involves conjugating glycans with diverse molecules through a linker. Valuable in glycan microarray studies, it enables controlled interactions with biomolecules, advancing research in glycobiology.

Ionic Liquid-Based Derivatization

Utilizing ionic liquids improves derivatization efficiency, especially in permethylation reactions. This innovative approach optimizes the process with high thermal stability and low volatility.

One-Pot Derivatization Strategies

One-pot strategies streamline glycan derivatization, reducing sample handling and improving overall efficiency. Practical for laboratories seeking simplicity and increased workflow efficiency.

Hydrazide Chemistry for Glycan Labeling

Hydrazide chemistry labels glycans selectively, facilitating quantification and structural analysis. Addressing challenges in isomeric glycan analysis, it provides specific labeling for accurate structure determination.

Isomeric Glycan Analysis through Derivatization

Derivatization for isomeric glycan analysis introduces tags to differentiate structures. This technique addresses challenges in distinguishing isomeric glycans, vital for studying complex glycan mixtures.

Quantitative Glycan Derivatization Strategies

Quantitative strategies introduce stable isotopes during derivatization for accurate glycan abundance measurement. Enhancing accuracy, this technique supports quantitative glycomics studies, aiding in understanding glycan dynamics in biological systems.

Overview of selected glycan derivatization approaches (Lageveen et al., 2022)

Applications of Glycan Derivatization

Fluorescence Labeling: Derivatization with fluorescent tags allows for sensitive detection of glycans in techniques such as high-performance liquid chromatography (HPLC) and capillary electrophoresis (CE). This is particularly useful for studying glycan composition and structure.

Mass Spectrometry Analysis: Glycan derivatization facilitates mass spectrometry analysis by improving ionization efficiency and providing enhanced sensitivity. Derivatized glycans can be analyzed using techniques like matrix-assisted laser desorption/ionization (MALDI) or electrospray ionization (ESI) mass spectrometry.

Glycan Profiling: Derivatization methods aid in glycan profiling, enabling the identification and quantification of various glycan species in complex biological samples. This is valuable for understanding glycomic changes associated with diseases or biological processes.

Chromatographic Separation: Derivatization enhances the chromatographic separation of glycans, making it easier to resolve individual glycan structures. This is particularly important in techniques like HPLC, where derivatization improves resolution and peak shapes.

Glycan Microarray Studies: Derivatization is employed in the creation of glycan microarrays for high-throughput screening of glycan-protein interactions. This is useful in the study of glycan-binding proteins and their roles in various biological processes.

Glycoprotein Analysis: Derivatization aids in the analysis of glycoproteins by improving the detection and characterization of glycan moieties attached to proteins. This is crucial in understanding the role of glycosylation in protein function.

Glycan Modification Studies: Derivatization is used to study glycan modifications, such as sialylation or fucosylation, providing insights into the functional diversity of glycans and their impact on cellular processes.

Glycan Structural Elucidation: Derivatization techniques contribute to the structural elucidation of glycans, allowing researchers to determine linkage patterns, anomeric configurations, and branching structures.

Case. Comparative Analysis of Glycan Derivatization Methods for Improved Mass Spectrometry Quantification in Glycomics Studies

Background:

Glycomics research involves the study of complex carbohydrate structures (glycans) and their roles in biological processes. Mass spectrometry (MS) is a powerful tool for glycan analysis, but the choice of derivatization method can significantly impact quantification accuracy. This study aims to compare different derivatization methods for glycans and assess their performance using nanoLC-MS.

Samples

The study utilized glycoprotein mixtures consisting of RNase B, fetuin, and IgG standard. Various derivatization methods were applied to release and label N-glycans, including PNGase F digestion, reductive amination labeling (2-AB, ProA), aminoxyTMT labeling, and reduction with permethylation. Different glycoprotein standards were employed to evaluate method performance.

Technical Method

• Enzymatic Release of Glycans: N-glycans were released from glycoproteins using PNGase F digestion.
• Reductive Amination Labeling: 2-AB and ProA labeling involved reductive amination under specific conditions.
• AminoxyTMT Labeling: AminoxyTMT labeling was performed following a recently published protocol, including dissolution, drying, and quenching steps.
• Reduction and Permethylation: Reduction with borane ammonium complex followed by solid-phase permethylation for reduced glycans.
• Glycosylamine Derivatization: RFMS labeling without purification after glycan release using Rapid PNGase F, RapiFluor-MS Reagent, and HILIC solid phase extraction.

Results

• Sample Preparation Time: RFMS derivatization exhibited the fastest sample preparation time due to the Rapid PNGase F release method.
• MS Intensity Enhancement: RFMS-labeled glycans showed the highest intensity, especially for neutral glycans, indicating superior signal enhancement.
• Sialylated Glycans: Permethylation demonstrated advantages in enhancing signals from acidic, sialylated glycans compared to other methods.
• NanoLC-MS Performance: NanoLC-MS analysis on a C18 column revealed differences in retention times and isomeric separation for reducing end-labeled and permethylated glycans.
• Adduct Formations: Different derivatization methods resulted in varied adduct formations and charge state distributions, impacting quantitative reliability and fragmentation patterns.

EIC of differently derivatized glycans

References

1. Lageveen‐Kammeijer, Guinevere SM, et al. "High sensitivity glycomics in biomedicine." Mass spectrometry reviews 41.6 (2022): 1014-1039.
2. Zhou, Shiyue, et al. "Direct comparison of derivatization strategies for LC-MS/MS analysis of N-glycans." Analyst 142.23 (2017): 4446-4455.