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Creative Biolabs is a forward-looking company as well as a leading custom service provider in the field of glycoprotein analysis. Based on our extensive experience and advanced platform, we have won a good reputation among our worldwide customers for successfully accomplishing numerous challenging projects in glycosylation site mapping. We are glad to help you get milestone success in your glycoprotein structure analysis project.
Glycosylation site mapping helps the identification of a glycosylation site, which can provide an indication of the function of that glycan. In addition, site-specific glycosylation analysis and assignment of a certain structure to a specific site can illuminate the protein’s glycosylation profile, microheterogeneity, and its activity. Glycosylation has great importance in the development of biological drugs because their glycan chains markedly affect product stability, activity, antigenicity, and pharmacodynamics. Glycosylation site mapping mainly contributes to glycan microheterogeneity.
Fig.1 Glycosite occupancy and heterogeneity in bovine lactoferrin (Hua, 2013).
N-glycosylation usually occurs on the asparagine residue of proteins that shuttle via the secretory pathway. N-linked glycosylation mapping characterizes the site-specific N-glycosylation including N-glycosylation site occupancy and site-specific glycan structure, which is important for the understanding of glycoprotein biosynthesis and function. Through glycosylation mapping, N-glycosylation site quantification can also be identified, helping reveal the critical role of macroheterogeneity in a variety of biological properties. There are a series of techniques we can provide to perform N-linked glycosylation mapping.
Technologies for N-linked glycosylation mapping in Creative Biolabs including but not limited to:
Reversed-phase high-performance liquid chromatography (RP-HPLC) can analyze the resulting peptide and glycopeptides fragments after digestion of a glycoprotein with specific proteases after reduction and alkylation. RP-HPLC combines on-line mass spectrometry to form a useful tool - RP-HPLC-MS. This method is semi-quantitative because different glycopeptides will ionize differently in a mass spectrometer.
Liquid chromatography-electrospray ionisation mass spectrometry (LC-ESI-MS) is a hyphenated mass spectrometry technique that combines the resolving power of HPLC separation with high mass accuracy of a mass spectrometer. It provides unparalleled sensitivity and selectivity for detection and quantification of site-specific glycan structure.
Solid-phase extraction using hydrophilic interaction liquid chromatography mass spectrometry (HILIC-SPE-MS) has become increasingly popular in the glycosylation site mapping because HILIC-SPE-MS is more selective for glycosylated species and is not biased toward particular glycan types. Furthermore, using that technique requires no chemical alteration of glycans.
Electrospray-ionization, collision-induced dissociation, tandem mass spectrometry (ESI-CID-MS/MS) allows for fragmentation of glycan species to be observed predominantly at the glycosidic linkages while the peptide backbone remains intact.
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. Determining occupied O-linked glycosylation sites is generally much more difficult than site-specific identification of N-linked glycosylation. There is no known amino-acid consensus sequence for O-linked glycans, and there is normally significant heterogeneity with O-glycosylated protein regions, both in the number of glycans and the extent of their occupancy. There are also a number of approaches that are based on the signal intensity of glycopeptide/peptide ions for O-linked glycosylation mapping, which is consistent with N-linked glycosylation mapping.
Technologies for O linked glycosylation mapping in Creative Biolabs including but not limited to:
Fig.2 Workflow of the glycosylation sites mapping (Ying, 2017).
Creative Biolabs is a leader in the field of glycoprotein analysis and has successfully completed a lot of therapeutic glycoprotein projects. We have experienced experts and advanced platforms that can provide excellent services for glycosylation site mapping. We offer a whole set of glycosylation site mapping 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.