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.

Core Technologies

Our company leverages state-of-the-art technologies to deliver highly accurate and sensitive relative quantification of site-specific glycoprotein glycosylation. Our core technologies primarily revolves around advanced mass spectrometry (MS) coupled with sophisticated chromatographic separation techniques.

• High-Resolution, Accurate-Mass (HRAM) MS
  We employ cutting-edge mass spectrometers, enabling unparalleled sensitivity and mass accuracy for the detection and characterization of intact glycopeptides. To achieve comprehensive glycopeptide characterization, we utilize a combination of fragmentation methods, including higher-energy collisional dissociation (HCD), electron transfer dissociation (ETD), and electron-transfer higher-energy collisional dissociation (EThcD).
• Liquid Chromatography (LC) for Glycopeptide Separation
  Our workflows incorporate advanced liquid chromatography systems, including nano-LC and ultra-high performance liquid chromatography (UCPLC), with various column chemistries such as C18, hydrophilic interaction liquid chromatography (HILIC), and porous graphitized carbon (PGC).

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.

Workflow

Applications

• Our service can be used as a highly specific biomarker to identify altered glycosylation patterns on specific proteins.
• Our service can be used to elucidate the precise role of individual glycosylation sites in protein function, stability, and cell interactions.
• Our services can be used to characterize the glycosylation of viral or bacterial surface proteins to understand immune escape mechanisms and design more effective vaccines.

Advantages of Us

• Our advanced MS and tailored workflows provide exceptional precision in identifying and quantifying glycosylation at individual amino acid residues. This level of detail is crucial for understanding the functional impact of specific glycan modifications.
• We employ optimized sample preparation protocols, highly efficient enrichment methods, and cutting-edge instrumentation to achieve high sensitivity, enabling the detection and quantification of even low-abundance glycopeptides.
• Our multi-fragmentation strategies (HCD, ETD, EThcD) coupled with diverse chromatographic separation techniques maximize the identification and characterization of a wide range of N-linked and O-linked glycopeptides, providing a more complete picture of the glycoproteome.

Publication Data

Frequently Asked Questions

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.

Associated Services

Protein Expression and Purification: To support your glycoproteomic studies, we provide high-quality protein expression and purification services, ensuring you have the pure protein samples required for accurate analysis.

Structural Elucidation: For a deeper understanding, we offer detailed structural analysis of individual glycans attached to your proteins, including monosaccharide composition, linkage analysis, and branching patterns.