Introduction
Unlike N-glycans, O-glycans are smaller, highly diverse branched-chain carbohydrate molecules that attach to
different protein structures. Site-specific O-glycosylation analysis is the key to exploring glycoprotein
structure-function relationships. It determines whether predicted O-glycosylation sites are modified and the
specificity of O-glycosylation at each of these sites. Glycosylation site analysis can not only intuitively obtain
information on glycosylation changes of many disease markers, but also make further sugar chain structure analysis
more direct and convenient. In recent years, mass spectrometry (MS)-based methods have proven to be the core
technology for site-specific O-glycosylation analysis due to their high resolution and sensitivity. The different
glycoforms typically released from proteins are identified as individual Glycans and their relative proportions, and
site-specific characterization of glycosylation was subsequently performed.
Fig. 1.
Schematic flowchart of sample processing for site-specific O-glycosylation analysis. (Yang S, et al., 2021)
Our Services
A number of features make the identification of O-linked sites difficult. 1) There is no known amino acid consensus
sequence for O-linked glycans. 2) There is often significant heterogeneity in O-glycosylated protein regions, both
in the number of glycans and in their degree of occupancy. Creative Proteomics utilizes
state-of-the-art MS platforms and other proteomic technologies to provide accurate and reliable O-linked
glycosylation site analysis services to identify genuine O-glycosylated peptides.
Creative Proteomics provides the following O-glycosylation site analysis service, including but not
limited to.
O-glycans are released by enzymatic or chemical treatment, followed by subsequent characterization.
- N-glycans are released with PNGase F and removed from solution.
- Use enzymes to digest proteins.
- O-glycopeptides are released by β-elimination.
- Enrichment of O-glycopeptides using HILIC (Hydrophilic Interaction Liquid Chromatography).
- O-linked glycosylation site occupancy was determined using LC-ESI-MS/MS.
Glycoproteins are digested into peptides and then glycan-attached peptides are analyzed.
- Digestion: Proteins are digested with proteases, usually trypsin, after reduction and alkylation. Additionly,
our scientists sometimes use several proteases for enzymatic digestion, thus increasing coverage of O-glycan
sites.
- Enrichment: Enriched with Amide-80, MAX or SAX SPE, respectively.
- Detection: Glycopeptides are direct analysis by LC-MS/MS.
Advantages of Our O-Glycosylation Site Analysis Service
- Sensitive, high-throughput, and reproducible quantification of O-linked glycan site occupancy.
- Multiple technology platforms.
- Handling complex biological matrices such as cells and tissues.
- Detailed report: Comprehensive report with raw data, parameters, data interpretation and bioinformatics
analysis.
- 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
O-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
- Yang S, Wang Y, Mann M, et al. (2021) Improved online LC-MS/MS identification of O-glycosites by EThcD
fragmentation, chemoenzymatic reaction, and SPE enrichment. Glycoconj J. 38(2):145-156.
- Li X, Wilmanowski R, Gao X, et al. (2022) Precise O-Glycosylation Site Localization of CD24Fc by LC-MS
Workflows. Anal Chem. 94(23):8416-8425.
