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:

Antibody
Lectin affinity chromatography
Chemoenzymatic Strategy
Boronic acid
Hydrazide Chemistry
Hydrophilic interaction liquid chromatography (HILIC)
Size exclusion chromatography (SEC)
Strong cation exchanger (SCX)
Reversed phase High Performance Liquid Chromatography (RP-HPLC)
Titanium dioxide (TiO₂)

Glycopeptide analysis 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.

Ordering Procedure

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

References

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.
Dalpathado DS, Desaire H. Glycopeptide analysis by mass spectrometry. Analyst. 2008 Jun;133(6):731-8.
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.

* For Research Use Only. Not for use in diagnostic procedures.

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From Our Clients

"I recently used their proteomics service for a project analyzing protein interactions in yeast models. The team was very responsive and helped clarify the methodology they employed, which made me feel confident in the results. The data quality was solid, with clear identification of several key proteins involved in our study. Their thorough analysis enabled me to pinpoint specific interactions that I hadn't considered before, which significantly improved the direction of my research. I appreciate their professionalism and support throughout the process."

Sarah Thompson, University of California, Berkeley

"Our lab collaborated with them on a project studying cancer biomarkers. The proteomics analysis provided was detailed and focused, specifically highlighting the differential expression of proteins between healthy and tumor samples. Their clear explanations of the data helped my team understand the biological implications. I also appreciated their willingness to revise the reports based on our feedback, ensuring that we had everything we needed for our publication. This collaborative spirit was invaluable."

Emily Rodriguez, Stanford University

"Our lab worked with them on a project studying the effects of diet on gut microbiota using proteomics. They used a label-free quantification method to analyze proteins in fecal samples before and after dietary intervention. The results showed significant changes in protein expression linked to microbial activity. This was pivotal for our hypothesis about diet-microbiota interactions. The clarity of their data presentation made it easy for our team to integrate these findings into our ongoing research."

Dr. Lisa Wong, University of Toronto

"My experience with Creative Proteomics during the mass spectrometry analysis was excellent. We sent in human saliva and mouse brain tissue samples, which they expertly analyzed using both LC-MS and GC-MS techniques. The results were invaluable, revealing key metabolites in the saliva and identifying biomarkers linked to brain function in the brain tissue."

Dr. Emily Carter, Senior Research Scientist

"The overall service from Creative Proteomics was outstanding. They made the entire process seamless and efficient, allowing us to focus on our research. We worked with leaf and root samples from various Arabidopsis genotypes for targeted metabolomics analysis. Their thorough profiling of primary and secondary metabolites gave us important insights into how the plants respond metabolically to environmental stress."

Dr. Laura Henderson, Plant Physiologist

"We had a pleasant collaboration with Creative Proteomics on mass spectrometry analysis of lipids. They conducted a detailed analysis of lipid species, providing us with important insights into lipid metabolism and its relationship with metabolic syndrome disease states."

Dr. Sarah Mitchell, Research Scientist

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