Glycoproteins are involved in a variety of biological processes, but glycoprotein analysis is a big challenge because of the low abundance and heterogeneity of glycans. As a forward-looking company as well as a leading customer service provider, Creative Biolabs has successfully explored innovative and versatile glycoprotein analysis platforms to provide diverse portfolio of glycoprotein structure analysis services. Glycan profiling is an important part of glycoprotein structure analysis we provide and glycan profiling service has helped us win a good reputation among our clients. We are glad to help you get milestone success in your glycoprotein project.
As a fundamental post-translational modification of proteins, glycosylation plays an important role in many physiological and pathological processes and is involved in multiple diseases, including cancer and autoimmune diseases as well as in congenital disorders of glycosylation. Glycoprotein structure analysis allows characterization of important biological molecules and would be of great diagnostic and clinical importance. Glycan profiling is an important part of glycoprotein structure analysis. As a novel approach to the structural analysis of glycans, glycan profiling put the emphasis on capturing essential information of target glycans in a rapid, sensitive, and high-throughput manner rather than defining covalent structures in a rigorous and time-consuming manner. Glycan profiling is particularly important for structural glycomics with the absence of a usefully automated sequencer analogous to protein and DNA sequencers. N-glycan and O-glycan profiling are two common types of analysis.
N-linked oligosaccharide is usually attached to the asparagine residue and N-glycosylation occurs solely on proteins that shuttle via the secretory pathway. With the increasing application of protein therapeutics, such as monoclonal antibodies, glycoproteins, hormones, cytokines and clotting factors in clinical practice, routinely N-glycan profiling has gained increasing scientific interest. At present, N-glycan profiling has been widely used in the identification of various diseases, including cancer, Alzheimer’s, and diabetes. Technologies for N-Glycan profiling in Creative Biolabs including but not limited to:
Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is a high-throughput method to analyze the structural characterization of glycosylated compounds with high sensitivity and robustness. It also prefers to profile mixtures of N-glycans due to the preference for singly charged ion formation that makes MALDI a better choice than electrospray ionization. During the detection, N-glycans are released by enzymatic deglycosylation (Peptide N-glycosidase A or F) or chemical methods such as Beta-elimination and hydrazinolysis, and then, they are analyzed after permethylation.
Ultra-high performance liquid chromatography linked with fluorescence detection/mass spectrometry (UPLC-FLD/MS) has been a powerful technology for N-glycan profiling. Glycoprofiling of enzymatic glycan release with PNGase F followed by derivatization by 2-aminobenzamide (2-AB) is a fast and robust technique to analyze larger numbers of samples with well-known N-glycan profiles.
High-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) is a sensitive and specific analytical method for the monosaccharide composition analysis. This direct analysis allows individual glycan monosaccharides analysis, providing not only the total glycosylation of a protein but also the amounts of specific monosaccharides.
Fig.1 Workflow of isolation and characterization of N-glycans from antibodies (Tayi, 2015).
O-acetylgalactosamine (O-GalNAc) and O-acetylglucosamine (O-GlcNAc) are the common O-glycosylations types, among which, O-GalNAc is attached to the hydroxyl group of the protein serine or threonine residues through an α-linkage, while O-GlcNAc is attached through a β-linkage. Unlike N-glycans, there is no known O-linked amino acid consensus sequence yet.
Technologies for O-Glycan Profiling in Creative Biolabs including but not limited to:
MALDI-TOF MS is the most common method for O-glycans profiling. Before O-glycans profiling, permethylation of the O-glycan to transform all hydroxyl groups into methyl ethers and stabilizes sialic acids by methyl esterification of their carboxyl groups is necessary, which allows MALDI-TOF MS to analyze of all O-glycans in the positive mode. Direct infusion of O-glycans of permethylation into the mass spectrometer can provide comprehensive information about glycans composition.
HPAEC-PAD can also provide details about fucosylation, which can impact protein function and signaling, gives an indication of the presence of O-linked glycans.
Fig.2 O-Glycan profiling.
Glycan profiling has become a regular study in glycosylation engineering, which is beneficial for the assignment of distinct functional properties to defined structural features. With years of experience, Creative Biolabs has successfully completed a lot of glycan profiling projects. We can offer a whole set of glycoprotein structure analysis service to help you get landmark development. We can also customize our offering to meet your specific project needs. If you are interested, please contact us without hesitation.