Protein glycosylation is a post-translational modification that may alter a protein's structure or function, which may be directly involved in the formation of diseases or altering the pharmacokinetics via stabilizing the protein conformation, improving solubility or protecting it from proteases. Therefore, it is very important to investigate glycan composition, site occupancy rates, and glycan structure to ensure protein structural integrity during drug development. Structural complexity is the main challenge in the analysis of protein glycosylation, which arises from a combination of these factors: the complex, branched structure of glycans, multiple glycan structures attached to each site, and multiple glycosylation sites per protein.
Figure 1. The complex and heterogeneous structure of glycans.
At Creative Proteomics, we provide glycan structural characterization analysis including residue composition, linkage types, attachment to aglycones, and three-dimensional (3D) glycan structure analysis with a combination of methods, including mass spectrometry (MS), nuclear magnetic resonance (NMR) spectroscopy, and high-performance liquid chromatography (HPLC), etc. This service is dedicated to investigating the nature and order of monosaccharide residues, the configuration and position of glycosidic linkages, and the nature and location of non-glycan entities to which they are attached.
Monosaccharide composition analysis provides estimated molar ratios of individual monosaccharide components of glycans and suggests the presence of specific oligosaccharide classes, such as N-linked glycans and O-linked glycans. This analysis involves several steps: cleavage of glycosidic linkages, fractionation of monosaccharides, detection, and quantification by MS.
Linkage analysis determines linkage positions based on the introduction of a stable substituent onto each free hydroxyl group of the native glycan, followed by cleavage of glycosidic linkages, producing partially methylated monosaccharides with a free hydroxyl group and a deuterium atom at C-1 at the linkage sites. The free hydroxyl groups are then acetylated resulting in partially methylated alditol acetates (PMAAs) that can be identified by a combination of electron impact (EI)-MS and GC retention times. Creative Proteomics provides linkage analysis for both N-glycan and O-glycan.
Most of the glycans do not have a well-defined three-dimensional structure in solution due to the inherent flexibility of glycosidic linkages. Nuclear overhauser effect (NOEs) can guide the construction of 3D structural models using tools such as the GLYCAM package. Full characterization of glycan conformation and dynamics can be further refined with molecular mechanics or molecular dynamics packages, or by incorporating NOEs with other NMR measurements, such as residual dipolar couplings and paramagnetic effects.
We work with a range of sample sources as follows.
As one of the leading companies in the proteomics field with years of experience, Creative Proteomics provides a full range of glycomics analysis service customized to your needs, including glycan profiling, site occupancy analysis, linkage analysis, structural characterization, glycopepetides analysis, glycan-related microarray assay, and polysaccharide analysis. Please feel free to contact us for discussing your project.
1. Mulloy B, Hart G W, Stanley P. Structural analysis of glycans//Essentials of Glycobiology. 3nd edition. Cold Spring Harbor Laboratory Press, 2017.