At CD BioGlyco, we leverage over many years of specialized expertise to provide state-of-the-art glycan profiling services, offering unparalleled insights into the intricate world of glycobiology. Our advanced platforms and highly skilled scientific team are dedicated to supporting your research and development initiatives, from fundamental discovery to preclinical research.

Overview

Similar in concept to the genome or proteome, glycome is a complete set of glycans and glycoconjugates generated by a cell or organism under specific conditions. Unlike amino acids in proteins or nucleotides in DNA, the monosaccharide units in glycan chains can be interconnected in many distinct ways, so there is no direct template or standard pattern for their biosynthesis. In addition, the structural diversity of oligosaccharide branches, the increased diversity of complex terminal saccharides, and various chemical modifications such as acetylation, phosphorylation, and sulfation, further increase the structural complexity of glycans. These great structural diversities enable glycans to encode a wide range of information, including cellular signaling, molecular recognition, and immune responses.

Identifying and analyzing the structure, quantity, and function of glycans is challenging. However, in recent years, this task has become easier with the advancement of science and technology and the continuous efforts of researchers. A variety of high-resolution and highly sensitive methods are now available, including mass spectrometry-based methods, high-performance liquid chromatography (HPLC), and lectin-based technologies.

Key Technologies

• Hydrophilic interaction liquid chromatography (HILIC): This is the most common method for separating released glycans. Glycans are polar molecules, and HILIC works by using a polar stationary phase and a non-polar mobile phase. The glycans interact with the polar stationary phase, and their separation is based on their polarity, with more polar glycans eluting later.
• Reverse-phase liquid chromatography (RPLC): The stationary phase is non-polar, and the mobile phase is polar. Separation is based on hydrophobicity, with more hydrophobic glycans eluting later.
• Ion-exchange chromatography (IEC): This technique separates glycans based on their charge. It's particularly useful for analyzing glycans that contain charged sugar residues, like sialic acids.
• Mass spectrometry (MS): After separation, the glycans are introduced into a mass spectrometer, which is the primary tool for identifying and characterizing them. MS provides highly precise information on a glycan's molecular mass and composition.

Advanced Glycan Profiling Services at CD BioGlyco

At CD BioGlyco, our carefully designed glycan analysis workflow encompasses multiple key stages, from sample preparation to data analysis, to ensure high-quality and reproducible results. Each step is optimized to maximize glycan recovery and analytical precision. Our process is as follows:

• Sample Preparation and Glycan Release

We utilize a variety of methods to isolate glycoproteins from serum, cells, tissues, or recombinant proteins. Glycans are then released from their protein backbones using highly specific enzymatic methods (e.g., PNGase F for N-glycans, O-glycosidase for O-glycans) or chemical methods (e.g., reductive β-elimination for O-glycans).

• Glycan Labeling and Purification

The released glycans are derivatized with fluorescent labels (e.g., 2-AB, 2-AA) or MS tags to enhance detection sensitivity. Following labeling, excess reagents and impurities are removed using methods such as solid-phase extraction (SPE) to ensure that only labeled glycans are analyzed.

• Separation and Detection

Separation is performed using high-performance liquid chromatography (HPLC) or ultra-high-performance liquid chromatography (UHPLC) systems. The choice of column and mobile phase depends on the specific glycan type and complexity to ensure optimal resolution of individual glycan structures.

• Data Acquisition and Analysis

MS and fragmentation data are collected and processed using bioinformatics software to identify and quantify individual glycan structures.

Workflow

Case Study

To address the challenges of analyzing complex glycoproteins, CD BioGlyco successfully developed an integrated, dual-mode analytical workflow that enables the concurrent characterization of both N- and O-glycans from a single sample pool. In this study, samples were efficiently homogenized and reduced to expose all glycosylation sites. Utilizing a precise sequential isolation strategy, N-glycans were first enzymatically released using PNGase F and separated from the remaining O-glycoproteins via advanced column purification, while the isolated O-glycoprotein fraction was subsequently subjected to reductive chemical elimination and permethylation to liberate and stabilize the O-glycans.

For downstream high-resolution structural elucidation, the two glycan fractions were analyzed using distinct, optimized MS platforms: native N-glycans were characterized on an MS in negative ion mode, while permethylated O-glycans were profiled on a MALDI-TOF-MS in positive ion mode. Finally, comprehensive data interpretation was executed using data software. This streamlined approach delivered an exhaustive, high-fidelity glycan map with precise structural and branching information, proving to be an invaluable tool for protein characterization.

Fig.1 Deconvoluted ESI-MS of sample N-glycans. (CD BioGlyco)

Table 1 Results of N-glycan identified. (CD BioGlyco)

Table 2 N-glycan feature distribution. (CD BioGlyco)

Publication Data

Applications

• Our team of experienced scientists with deep expertise in glycan profiling ensures meticulous analysis and in-depth interpretation of your data.
• Our state-of-the-art instrumentation ensures exceptional sensitivity, accuracy, and reproducibility when processing samples.
• From sample preparation to data analysis, we adhere to strict procedures at every stage of the workflow, ensuring consistent and reliable results.
• Our services are highly customizable; whether you require high-throughput screening or in-depth structural elucidation, we tailor experimental designs to meet your specific research goals and sample requirements.

Advantages

• Glycan profiling services are used to monitor the consistency and efficacy of the production of therapeutic glycoproteins, including monoclonal antibodies and vaccines.
• Glycan profiling services are used to analyze the role of glycans in intercellular communication, immune regulation, and developmental processes.
• Glycan profiling services are crucial for understanding pathogen glycan structure and designing effective immunogens.
• In food and nutritional science, glycan analysis helps understand the bioactivity of prebiotics and probiotics, as well as the nutritional value of various food ingredients.

Frequently Asked Questions

At CD BioGlyco, our comprehensive glycan profiling services are backed by extensive expertise and advanced technologies designed to accelerate your research discoveries and support your development goals. Please contact us today to discuss how our tailored solutions can help your specific project.

Associated Services