Glycopeptides Analysis

What is Glycoprotein?

The peptide chain of a protein can be covalently attached to one or more carbohydrate chain, forming a glycoprotein or glycopeptide. The process of glycosylation is one of the most prevalent and crucial post-translational modifications in proteins, playing a pivotal role in regulating the properties and functions of a diverse array of proteins, such as cellular immunity, signal transduction, protein translation regulation, and protein degradation. According predict, half of proteins in mammalian cells can be glycosylated and almost all membrane and secreted proteins contain glycosylation modifications. The association between aberrant glycosylation and human diseases has been extensively documented in numerous reports. Protein glycosylation can be primarily categorized into two types: N-glycosylation and O-glycosylation. N-glycosylation refers to the attachment of glycans to the asparagine (N) residue in proteins starting with GlcNAc, following a consensus sequence of N-X-S/T, where X represents any amino acid except proline. For O-glycosylation, glycans are covalently attached to the serine (S) and threonine (T) residues starting with GalNAc. This modification is also highly dynamic and actively engages in crosstalk with other modifications, notably phosphorylation. However, (i) glycosylation is normally of low stoichiometry in cells, (ii) the heterogeneity and complexity of glycan structure, making it difficult to pinpoint the glycosylation sites and to deconvolute the glycan structures, therefore, it is critical to effectively enrich glycopeptides/glycoproteins prior to analysis.

With the advancement of mass spectrometry (MS) instrumentation and glycoproteomic methodologies, it is now possible to perform global and site-specific characterization of protein glycosylation for comprehensive characterization of glycoproteins in intricate biological samples has become feasible. Importantly, when combined with quantitative glycoproteomic methods, the abundance of glycoproteins/glycopeptides in different samples can be accurately quantified. This enables the investigation of how glycosylation impacts protein properties and functions, as well as facilitates the identification of abnormal glycosylation events associated with human diseases.

Figure 1. Schematic diagram of the steps involved in glycopeptide analysis ^[2]

Glycopeptide Analysis in Creative Proteomics

Currently glycopeptide analysis commonly employs a bottom-up proteomics approach. This method typically involves a combination of specific enzymatic proteolysis followed by fractionation of glycopeptides by affinity chromatography or liquid chromatography and eventually glycopeptide analysis by MS. We have command various strategies to enhance the enrichment of Glycopeptides for comprehensive identification, and we have also developed multiple quantification methods including SILAC, iTRAQ/TMT, label-free, DIA.

Methods for Glycopeptides enrichment:

1. Antibody
2. Lectin affinity chromatography
3. Chemoenzymatic Strategy
4. Boronic acid
5. Hydrazide Chemistry
6. Hydrophilic interaction liquid chromatography (HILIC)
7. Size exclusion chromatography (SEC)
8. Strong cation exchanger (SCX)
9. Reversed phase High Performance Liquid Chromatography (RP-HPLC)
10. Titanium dioxide (TiO₂)

Figure 2. A streamlined pipeline for multiplexed quantitative site-specific N-glycoproteomics ^[3]

With over a decade of experience and a specialized research team in glycopeptide analysis, Creative Proteomics possesses comprehensive analytical capabilities for glycopeptides, including intact glycopeptide analysis as well as specific analyses for N-glycosylation or O-glycosylation. The deep coverage of intact glycopeptides can be easily achieved using a cost-effective method, which involves typical peptide preparation combined with high-resolution MS. The high specificity glycopeptide enrichment approaches can be combined to meet the increased demand for glycopeptide identification. For specific O- or N-glycosylation analysis, we can utilize PNGase F to release N-linked glycans from glycoproteins or chemical reagent to release O-linked glycans from glycoproteins.

Applications of Glycopeptide Analysis

1. Analysis of glycosylated proteome changes and signaling pathways under different experimental conditions.

2. Candidate glycoprotein marker screening.

3. Glycans content analysis of individual glycoproteins, glycans structure confirmation and quantitative analysis.

4. Antibody research.

Technologies platform

1. Professional detection and analysis capability: Equip specialized proteomics, quantification proteomics and bioinformation research team, strict quality control system, together with ultra-high resolution detection system and professional data pre-processing and analysis capability, ensure reliable and accurate data.

2. High specificity: Optimization of experimental design and methods, N-Link Glycan Search, O-Link Glycan Search, or Glycan Search: N-Link+ O-Link.

3. High stability and reproducible: Obtain consistent and reproducible inter- and intra- assay results for data analysis.

4. High resolution and sensitivity: Triple TOF 5600, Q-Exactive, Q-Exactive HF, Orbitrap Fusion Tribrid™, etc.

5. High selectivity: We can provide a wide range of multi-technological services and efficiently handle various types of samples, while remaining cost-effective and ensuring short turnaround times for your projects.

Samples Requirement

We can accept a variety of samples, including but not limited to:

Tissue: animal tissue > 50 mg

Plant tissue > 100 mg

Cell: suspension cell > 1 ×10⁷

adherent cell > 1 ×10⁷

microorganism > 100 mg or 5 × 10⁷ cells

Body fluid: serum/plasma > 100 uL

Protein: total protein >300 μg and concentration >1 μg/μL.

Note: In order to ensure the test results, please inform the buffer components if you give us proteins, whether it contains thiourea, SDS, or strong ion salts. In addition, the sample should not contain components such as nucleic acids, lipids, and polysaccharides, which will affect the separation effect.

Results Delivery

1. Detailed report, including experiment procedures, parameters, etc.

2. Data analysis results and raw data.

How to place an order

As one of the leading companies in the omics field with over years of experience in omics study, Creative Proteomics offers a diverse range of cutting-edge technologies for glycopeptides analysis research, facilitating precise quantification of glycoprotein levels and accurate identification of glycosylation sites. Please feel free to contact us by email to discuss your specific needs. Our customer service representatives are available 24 hours a day, from Monday to Sunday.

*If your organization requires signing of a confidentiality agreement, please contact us by email.

References

1. Xu S, Xu X, Wu R. Deciphering the Properties and Functions of Glycoproteins Using Quantitative Proteomics. Journal of Proteome Research. 2023 Jun 2;22(6):1571-1588.
2. Dalpathado DS, Desaire H. Glycopeptide analysis by mass spectrometry. Analyst. 2008 Jun;133(6):731-8.
3. Fang P, Ji Y, Silbern I, et al. A streamlined pipeline for multiplexed quantitative site-specific N-glycoproteomics. Nature Communications. 2020 Oct 19;11(1):5268.