Introduction
Protein glycosylation is the most diverse and complex form of protein modification, greatly increasing protein
heterogeneity to promote functional plasticity. Compared to N-linked glycosylation, the study of O-linked
glycosylation is one of the most challenging post-translational modification analyses due to the structural
complexity of added glycans and the technical challenges to unambiguously characterize them.
The process of O-linked glycosylation links different O-linked glycans to Ser and Thr residues, or the less common
Tyr residues in the human proteome. Notablly, O-linked N-acetylgalactosamine (O-GalNAc) additions were the
predominant type. Definitive characterization of O-linked glycoproteins requires quantitative analysis of O-linked
glycosylation sites and their corresponding glycans. Unlike N-glycosylation, O-glycosylation is not governed by the
presence of a linear consensus sequence in the amino acid chain, so O-glycosylation can't be predicted from
linear amino acid sequences.
Fig. 1. Current Methods
for the Characterization of O-Glycans. (Wilkinson H, et al., 2020)
Our Services
Liquid chromatography fluorescence analysis is only suitable for the analysis of unreduced labeled glycans, but both
unreduced and unlabeled reduced glycans are suitable for mass spectrometry detection. Therefore, Creative
Proteomics offers two commonly used MS techniques for the characterization of O-glycans, including
matrix-assisted laser desorption/ionization (MALDI) MS and electrospray ionization (ESI) MS.
At Creative Proteomics, our high-quality laboratory technicians analyze O-glycans by using MALDI-TOF
MS or (ESI) MS. Permethylation of the O-glycan to transform all hydroxyl groups into methyl ethers and stabilizes
sialic acids by methyl esterification of their carboxyl groups is necessary, which allows MALDI-TOF MS to analyze of
all O-glycans in the positive mode.
Experiment Process
Fig. 2. Workflow for O-glycan profiling
service.
- Protein denaturation.
- Enzymatic removal of N-glycans (PNGAse F).
- Release: O-linked glycans are typically released by reductive alkaline β-elimination.
- Isolation: The released O-glycans can be enriched by hydrophilic interaction chromatography (HILIC).
- Labeling: To improve high performance liquid chromatography (HPLC) and MS based glycan analysis, permethylation
of the released O-glycans to convert all hydroxyl groups to methyl ethers and stabilize sialic acid by methyl
esterification of its carboxyl groups.
- Seperation: Labeled o-glycans were enriched by IHLIC.
- Detection: Enriched O-glycans are then detected by MALDI-TOF MS (matrix-assisted laser
desorption/ionization-time of flight mass spectrometry) or ESI-MS (electrospray ionization mass spectrometer).
- Report.
*Note: Creative Proteomics can also perform glycan studies at the glycopeptide or glycoprotein
level, providing you with complete O-glycan characterization services for glycosylation.
Advantages of Our O-Glycan Analysis Service
- High-throughput, reproducible O-glycan patterns with sensitivity and specificity.
- Process a variety of complex biological matrices such as cells and tissues.
- High coverage of O-glycans.
- Fast turnaround time.
- One-stop service for fully customized design.
- Detailed reports include experimental procedures, LC and mass spectrometer parameters, MS raw data files,
O-glycan analysis results, and bioinformatics analysis.
ICH Q6B guidelines require comprehensive characterization of glycosylation of glycoproteins. With years of experience
and an experienced scientific team, Creative Proteomics provides high-quality O-glycan analysis
services. Additionally, we can provide fully custom project designs to meet any specific requirements. If you are
interested, please contact us or send us an inquiry directly.
References
- Wilkinson H, Saldova R. (2020) Current Methods for the Characterization of O-Glycans. J Proteome Res.
19(10):3890-3905.
- Zhang L, Luo S, Zhang B. (2016) Glycan analysis of therapeutic glycoproteins. MAbs. 8(2):205-215.
