Understanding the function of glycoproteins and their changes associated with the disease is complicated by challenges related to analysis and characterization. CD BioGlyco is proficient at relative quantification of glycoproteins at site-specific glycosylation levels to help researchers understand the pathogenesis of diseases.

Introduction

Protein glycosylation, the process by which glycans are transferred to proteins by the action of glycosyltransferases and form glycosidic bonds with specific amino acid residues on proteins, consists of a distribution of associated glycoforms at each glycosylation site. Since the biosynthetic substrate concentration and translocation rate are related to structure and other aspects of cellular phenotypes, site-specific glycosylation cannot be accurately predicted from the information of genomic, transcriptomic, or proteomic. Rather, it is critical to relative quantify glycosylation at each specific protein site to provide information on disease mechanisms.

At present, well-established mass spectrometry-based methods allow for relative quantification of glycosylation sites and glycan composition of singly glycosylated proteolytic peptides. To make such quantitative comparisons, it is essential to sample glycosylation distributions with accuracy and sufficient coverage to provide a reliable assessment of glycosylation changes occurring in biological cohorts.

Fig.1 Site-specific glycoforms of sialidase treated hemopexin (HPX) and complement factor H (CFH) separated by RP chromatography and detected by MRM-MS. (Goldman, 2015)

Our Services

CD BioGlyco provides a variety of technology platforms for the relative quantification of glycoprotein at the site-specific glycosylation level. The strategies we provide include but are not limited to:

• N-linked site mapping.
• Use endo-F or H enzymes to release the N-glycans and leave a GlcNAc core attached to the glycopeptide. Then mapping the position of the remaining GlcNAc by LC-MS allowed us to obtain information on site location as well as site occupancy.
• The asparagine residues at the N-glycosylation site of the glycopeptide are converted to aspartic acid by enzymatic deglycosylation, which increases the mass of the peptide on the N-glycosylation site thereby enabling recognition of the glycosylation site. However, confidence can be added by performing this deglycosylation reaction in "heavy" water (18O water), which increases the mass even more. The positions of the ¹⁸O-labeled aspartate residues are then mapped by LC-MS.
• O-linked site mapping by BEMAD. The reductive β-elimination of the O-glycan leaves a modified threonine or serine residue in the pre-glycopeptide. This residue is reactive to the thiol functional group and this property is utilized in the BEMAD method to label this residue for unambiguous MS detection.
• Multiple reaction monitoring (MRM), parallel reaction monitoring (PRM), selective reaction monitoring (SRM), and other mass spectrometry (MS)-based techniques such as LC-MS/MS-MRM are also effective approaches for quantifying site-specific glycosylation of glycoproteins.

Advantages of Us

• Constantly updated and upgraded technology platform
• Extensive experience in terms of relative quantification
• Powerful data analysis tools
• High accuracy and short experimental period

CD BioGlyco is a company with extensive experience and a good reputation in the field of glycobiology. Our advanced experimental platforms and well-trained researchers will greatly accelerate our clients' research in glycobiology. If you have a demand for relative quantification of glycoprotein, please contact us to obtain more information.

Reference

1. Goldman, R.; Sanda, M. Targeted methods for quantitative analysis of protein glycosylation. Proteomics Clinical Applications. 2015, 9(1-2):17-32.