Introduction
Protein N-glycosylation plays an important role in physiological and pathological processes. Characterizing
site-specific N-glycosylation, including N-glycan macroscopic heterogeneity (glycosylation site occupancy) and
microscopic heterogeneity (site-specific glycan structure), is important for understanding glycoprotein biosynthesis
and function.
N-glycosylation is a post-translational modification of proteins involved in a variety of physiological and
pathological processes. N-glycosylation site occupancy reflects the ratio of N-glycosylation in a protein, a
phenomenon that in turn reveals the availability of sites, substrate concentrations, and enzyme kinetics in the ER.
The properties of glycoproteins are largely affected by site occupancy, and defects in site occupancy may be
detrimental to physiological functions. Therefore, accurate determination of site occupancy is important
significance to fully understand the impact of protein glycosylation on human health.
Figure
1. MS-based labeling and label-free technologies for quantification of N-glycosylation site occupancy. (Zhang S,
et al., 2017)
Our Services
Liquid chromatography-mass spectrometry (LC-MS)-based quantification has been widely used and provides fairly
advanced quantification of N-glycosylation site occupancy in a sample. At Creative Proteomics, we
provide tools for the quantification of N-glycosylation sites for Labeled and label-free techniques for
glycosylation site occupancy. SILAC, TMT, dimethylation, and iTRAQ are commonly used to introduce "heavy"
and "light" isotope or isobaric labels into samples, while label-free methods are based on spectral
counting or peptide ionic strength.
Creative Proteomics provides the following N-glycosylation site analysis services, including but not
limited to.
- Label Quantification of N-Glycosylation Site Occupancy
A significant advantage of the labeling approach is the ability to efficiently analyze differentially labeled samples
simultaneously in a single run. We can offer trypsin-catalyzed 18 O, SILAC and iTRAQ labeling methods for
N-glycosylation site occupancy analysis. Isotope-encoded glycosylation site-specific tags (IGOTs) are available for
large-scale N-glycosylation site analysis.
A general strategy for the analysis of variable N-glycosylation sites in glycoproteins includes the following
procedures.
- PNGaseF enzymatic digestion: Protein n-glycosidase F (PNGase F) is usually used to separate polysaccharides from
proteins in heavy water. During this process, the originally glycosylated asparagine undergoes deamidation to
aspartic acid, increasing O18 retouch. Because of the change in molecular mass upon introduction of O18,
glycosylation sites can be detected by comparing aspartic acid-containing peptides with non-glycosylated
asparagine-containing peptides.
- Glycan release.
- Separation of Glycopeptides by Liquid Chromatography.
- Mass detection: C18-LC-MS/MS or MALDI-TOF-MS/MS analysis.
- Label-Free Quantification of N-glycosylation Site Occupancy
Label-free quantification of N-glycosylation site occupancy is usually based on peptide ion intensity or peak area.
Its significant advantage is the simple workflow.
A general strategy for the analysis of variable N-glycosylation site occupancy in glycoproteins includes the
following procedures.
- Protein digestion of proteins to produce peptides.
- Use PNGase F to release N-glycans: PNGase F specifically releases N-glycans linked to the nitrogen of Asn and
converts Asn residues to Asp with a mass gain of 0.984 Da.
- Enrichment of N-glycopeptides by HILIC (Hydrophilic Interaction Liquid Chromatography).
- Determination of N-linked glycosylation site occupancy using LC-ESI-MS/MS.
Advantages of Our N-glycosylation Site Analysis Service
- A variety of marking methods and technology platforms.
- Strict quality control management.
- Quick turnaround.
- One-stop service.
Report You Can Receive Includes:
- Experiment procedures.
- Parameters of liquid chromatography and mass spectrometer.
- Raw MS data files.
- Glycosylation site results.
- Bioinformatics analysis.
The ICH Q6B guideline requires comprehensive characterization of glycoprotein glycosylation. With years of experience
and an experienced scientific team, Creative Proteomics provides diverse and systematic
N-glycosylation site analysis services. Additionally, we can provide fully custom project designs to meet any
specific requirements. Our efficient service and accurate results save clients time and money. If you are
interested, please contact us or send us an inquiry directly.
References
- Zhang S, Li W, Lu H, et al. (2017) Quantification of N-glycosylation site occupancy status based on
labeling/label-free strategies with LC-MS/MS. Talanta. 170:509-513.
