Glycan Structure Analysis

Glycan Structure Analysis is a scientific process that allows for an in-depth understanding of glycans, their structure, modifications, as well as their roles in various biological systems. These complex carbohydrate structures, attached either directly to proteins (N-glycans and O-glycans) or lipids (glycolipids) play crucial roles in several biological processes. This includes cell signaling, molecular recognition events, and protein folding, making their analysis essential.

Glycan structure analysis involves a meticulous investigation of the monosaccharide composition, stereochemistry, size, sequence, and positioning of glycosidic linkages and specific moieties. By employing various analytical techniques and approaches, glycan structure analysis unravels the complex structure-function associations and paves the way for novel therapeutic developments.

Glycan Structure Analysis Solutions at Creative Proteomics

Monosaccharide Composition Analysis - Understanding the individual monosaccharide units and their quantities in a glycan structure.

Oligosaccharide Population Analysis - Detailing the different oligosaccharides derived from a glycoprotein, providing insights into their diversity.

Glycan Sequencing - Sequencing elucidates the arrangement of monosaccharide units in a glycan, revealing its structural uniqueness.

Glycan Linkage Analysis - Unraveling the types of linkages between monosaccharide units and their configurations which significantly influence glycan function.

Glycan Modification Analysis - Analyzing modifications like sulfation, phosphorylation, and methylation within the glycan structure that understanding their complex behaviors.

Techniques Employed in Glycan Structure Analysis

In Creative Proteomics, various advanced mass spectrometry-based techniques are used for glycan structure analysis:

Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry (MALDI-MS): An ideal tool for analyzing high-molecular-weight biomolecules, MALDI-TOF/TOF MS is adept at maintaining the integrity of the glycan structure during the ionization process and ensures accurate mass detection.

HPLC-ESI-MS (High-Performance Liquid Chromatography-Electrospray Ionization Mass Spectrometry): Used for the analysis of smaller and more complex glycans, HPLC-ESI-MS separates glycans based on their affinity towards different solvents followed by mass detection.

nanoLC-ESI-HCD MS/MS (Nano-Liquid Chromatography-Electrospray Ionization-Higher-energy Collisional Dissociation MS/MS): This technique offers a higher sensitivity for glycan detection. It involves separating the glycans followed by tandem mass spectrometry for detailed analysis.

Glycan Microarray Analysis: Creative Proteomics utilizes glycan microarray technology to interrogate the binding specificities of glycan-binding proteins (lectins) and antibodies. By immobilizing a diverse array of glycans onto solid supports, we can screen for interactions with target molecules, facilitating the identification of glycan-binding partners and elucidation of glycan-mediated biological processes.

Advantages of Glycan Structure Analysis

Experienced Team: Experienced specialists by your side for advice and support throughout the project.

Advanced Techniques: Utilization of advanced technology and techniques for precise and accurate analysis.

Customized Solutions: Project customization according to the unique requirements of each client.

Comprehensive Analysis: From the basics to advanced analysis, Creative Proteomics offers a comprehensive suite of glycan structure analysis.

High-quality Data: Provision of high-quality, reliable data that paves the way for further research and development.

Sample Requirements for Glycan Structure Analysis

Applications of Glycan Structure Analysis

Disease Biomarker Discovery: Aberrant glycosylation patterns have been implicated in the pathogenesis of numerous diseases, including cancer, autoimmune disorders, and infectious diseases. Glycan structure analysis facilitates the identification of disease-specific glycan biomarkers, enabling early diagnosis, prognosis, and monitoring of disease progression.

Biopharmaceutical Development: Glycosylation is a critical post-translational modification in biopharmaceuticals, influencing their efficacy, stability, and immunogenicity. Glycan structure analysis plays a pivotal role in characterizing glycosylation profiles of therapeutic proteins and monoclonal antibodies, ensuring product quality, consistency, and regulatory compliance.

Glycoengineering and Glycobiology: Understanding the structural features of glycans is essential for glycoengineering efforts aimed at modulating glycan structures for therapeutic purposes. Glycan structure analysis provides insights into glycan biosynthesis pathways, glycosyltransferase activities, and glycan-mediated signaling pathways, advancing our understanding of glycobiology and facilitating the development of novel glycotherapeutics.

Vaccine Development: Glycans serve as targets for vaccine development against pathogens such as bacteria and viruses. Glycan structure analysis enables the characterization of glycan epitopes involved in host-pathogen interactions, guiding the design and optimization of glycan-based vaccines with enhanced efficacy and specificity.

Nutritional and Food Science: Glycans are abundant in various food sources and play essential roles in nutritional quality, taste, and texture. Glycan structure analysis facilitates the characterization of food glycomes, providing insights into the composition and functional properties of food-derived glycans. This information is valuable for food quality control, nutritional labeling, and the development of functional food products.

Glycan-Targeted Therapeutics: Glycans are attractive targets for therapeutic intervention in various diseases, including cancer, infectious diseases, and inflammatory disorders. Glycan structure analysis aids in the identification of glycan-binding ligands, such as lectins and antibodies, for targeted drug delivery and immunotherapy applications.

Glycan-Protein Interactions: Glycans play crucial roles in mediating interactions between proteins and other biomolecules. Glycan structure analysis enables the characterization of glycan-binding specificities of proteins, facilitating the elucidation of glycan-protein interaction networks and their implications in cellular signaling, immune response modulation, and disease pathogenesis.

Case. Comprehensive Glycomic Profiling of Protein Glycosylation Using Permethylation and Tandem Mass Spectrometry

Background:

Glycosylation, a common protein post-translational modification, plays crucial roles in biological processes. Despite advancements in bioanalytical techniques, comprehensive profiling of protein glycosylation remains challenging due to the complexity of glycan structures, their labile nature, and the limitations of existing analytical methods.

Samples:

Glycoproteins, ribonuclease B, fetuin, IgG standard, and pooled human blood serum were utilized as samples in the study. These samples represent a diverse range of glycoproteins and biological sources, allowing for comprehensive glycan analysis.

Technical Method:

Glycan Release, Reduction, and Permethylation: Glycoproteins or blood serum samples were denatured and treated with PNGase F to release glycans. Glycans were then purified, reduced, and permethylated to stabilize their structures and enhance ionization efficiency.

LC-MS Methods: Glycans were separated using a HyperCarb PGC column coupled with an UltiMate 3000 nano UHPLC system. Mobile phases A and B were composed of water/formic acid and acetonitrile/water/formic acid, respectively. LC separation was followed by analysis using an LTQ Orbitrap Velos mass spectrometer.

Data Interpretation: Glycan compositions were identified using extracted ion chromatograms (EICs), and MS/MS scans were analyzed for structure elucidation using GlycoWorkbench software.

Results and Discussion: The study successfully elucidated glycan isomeric structures, including identification of core fucosylation and differentiation of galactose linkages, using LC-MS/MS. Permethylation mitigated fucose migration, while CID and HCD MS/MS techniques provided detailed structural information.

Results:

Identification of Core Fucosylation:

• Permethylation enabled the identification of core fucosylation sites.
• Diagnostic ions at m/z 468.27 distinguished core fucosylation from branch fucosylation.
• MS/MS spectra revealed fucose migration and rearrangement, addressed by permethylation.

Differentiation of Galactose Linkages:

• LC separation facilitated the identification of galactose linkages.
• Isomeric separation of galactose residues based on β 1,3 or β 1,4 linkages was achieved.
• CID and HCD MS/MS methods provided complementary fragmentation patterns for linkage determination.

Extracted ion chromatogram (EIC) for two reduced and permethylated F1A2G1 isomeric structures, [M + 2H]2+, at m/z 1017.5413 (a) and four reduced and permethylated F1A2G2S1 isomeric structures, [M + 3H]3+, at m/z 867.1213 (b)

CID MS/MS spectra (a, b) and HCD MS/MS spectra (c, d) for two reduced and permethylated fetuin triantennary glycan isomers, [M + 2H]2+, that only differ in the linkage of one terminal galactose residue

Reference

1. Zhou, Shiyue, et al. "LC-MS/MS analysis of permethylated N-glycans facilitating isomeric characterization." Analytical and bioanalytical chemistry 409 (2017): 453-466.