Glycomics Institute of Alberta

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Glycoprotein Workflow

So you think you have a glycoprotein? Oftentimes the first clue that you may have a glycoprotein are smears appearing during gel electrophoresis. This is due to the heterogeneity of attached glycans. Use our Glycoprotein Workflow to figure out what to do with it!

Some Considerations Before Starting

Glycan Complexity

Glycans are highly diverse in terms of structure and play critical roles in numerous biological processes. They can provide insights into disease mechanisms, biomarker discovery, and therapeutic target identification.

N- & O-Glycosylation

N- and O-glycosylation are the two major types of protein glycosylation. N-Glycosylation occurs at Asn in the Asn-X-Ser/Thr motif; O-Glycosylation attaches to Ser/Thr. Both affect protein folding, trafficking, stability, and interactions.

Heterogeneity

The approaches and techniques employed may vary depending on the experimental goals, resources available, and the glycoprotein under investigation. The workflow should be adapted based on your specific requirements.

Step 1 Glycan Release

Employ enzymatic or chemical methods on the purified glycoprotein to obtain released glycans and deglycosylated proteins.

Option 1. Enzymatic Treatment

N-Glycosylation
O-Glycosylation

Treat the glycoprotein with PNGase F, which specifically cleaves the N-glycosidic bond between the GlcNAc residue and the Asn residue in the Asn-X-Ser/Thr motif in N-glycosylation. If PNGase F treatment successfully removes the glycans, it indicates the presence of N-glycosylation.

Option 2. Chemical Treatment

N-Glycosylation
O-Glycosylation

Treat the glycoprotein with hydrazine in the presence of mild alkali in a process known as hydrazinolysis. Hydrazinolysis selectively cleaves the N-glycosidic bond, releasing the deglycosylated protein from the N-glycans.

Note: Glycans are not expected to remain intact during chemical treatment. These reactions break the glycosidic bonds between the sugar units, resulting in the release of individual monosaccharides or smaller oligosaccharide fragments. It will still be possible to perform monosaccharide composition analysis or quantification of specific sugar units.

Step 2 Separation of N- and O-glycosylation

Use SDS-PAGE to analyze the molecular weight shift of the glycoprotein before and after treatment. Glycosylation typically adds significant mass to the glycoprotein, so after deglycosylation, the protein band should show a decrease in molecular weight. By comparing the migration pattern of the protein before and after treatment, you can infer the successful removal of N- or O-glycans.

Alternatively, use Western blotting to assess the glycosylation status of proteins using glycan-specific antibodies or lectins. A decrease or absence of binding to glycan-specific antibodies or lectins after treatment suggests successful N- or O-glycan removal.

N-Glycosylation
O-Glycosylation

N-glycans are predominantly characterized by a common core structure consisting of a complex or high-mannose type glycan attached to the Asn residue in the Asn-X-Ser/Thr motif. For example, lectins that preferentially bind high-mannose structures, such as ConA or wheat germ agglutinin (WGA), would exhibit stronger binding to high-mannose N-glycans compared to complex-type N-glycans.

Step 3 Glycan Sample Preparation

Process the released glycans to ensure the purity and compatibility of the sample for subsequent analysis.

1. Desalting

  • Involves selectively binding glycans to a solid-phase material while allowing contaminants to be washed away, resulting in purified glycans that can be eluted for further analysis
  • Particularly effective for removing salts, proteins, small molecules, lipids, and other interfering substances
  • Involves selectively partitioning glycans from a sample into an organic solvent phase, separating them from the aqueous phase, and subsequently recovering the glycans for further analysis
  • Particularly effective for removing nonpolar impurities, such as hydrophobic compounds, lipids, or nonpolar solvents
  • Involves the separation of glycans from small molecules and salts by diffusion through a semi-permeable membrane based on their size and molecular weight
  • Particularly effective for removing low molecular weight compounds, ions, and buffer components

2. Derivatization (optional)

Derivatization is accomplished by chemically modifying the glycans through specific reactions with derivatizing agents. Some common derivatization techniques used in glycan analysis include fluorescent labeling, permethylation, amidation, and hydrazide chemistry.

Step 4 Glycan Analysis

Determine the composition, structure, and relative abundance of glycans using various techniques, such as including liquid chromatography (LC), mass spectrometry (MS), and capillary electrophoresis (CE).

1. LC-Based Analysis

  • Enables the separation and analysis of glycans based on their hydrophobic properties
  • Well-suited for the analysis of glycan structures with higher hydrophobicity, such as glycans containing fatty acid or lipid modifications
  • Enables the separation and analysis of glycans based on their polarity and hydrophilicity
  • Well-suited for the analysis of glycans with varying degrees of hydrophilicity, such as neutral, acidic, or sialylated glycans
  • Enables the separation and analysis of glycans based on their molecular size, providing information about the distribution and relative abundance of glycans with different sizes in a sample
  • Particularly effective for separating and analyzing high-molecular-weight glycans, such as polysaccharides and glycoconjugates

2. MS-Based Analysis

  • Glycans are co-crystallized with a matrix compound and then subjected to laser irradiation, resulting in desorption and ionization of the glycans
  • Provides information about glycan masses and relative abundances in the form of a mass spectrum
  • Can be coupled with tandem mass spectrometry (MS/MS) to obtain limited structural insight through fragmentation analysis
  • Involves ionizing glycans in solution through electrospray, followed by their transfer into the gas phase
  • Provides detailed structural information, confirms monosaccharide composition, allows for quantitative analysis to measure glycan abundance, and has the ability to differentiate glycan isomers

3. CE-Based Analysis

  • Involves separating and analyzing charged molecules, such as glycans or proteins, based on their size and charge using a capillary filled with a gel matrix and an electric field
  • Provides information about glycan composition, structural heterogeneity, and relative abundance in a sample
  • Involves separating and analyzing fluorescently labeled glycans using capillary electrophoresis and detecting them with laser-induced fluorescence
  • Provides information about glycan composition, structural heterogeneity, and relative abundance in a sample

4. ELISA

ELISA can be utilized to gain insights into the presence or levels of particular glycan structures. This can be achieved by coating ELISA plates with glycan-specific antibodies or lectins, and then detecting the binding of the glycoprotein sample. It can also be employed as a quality control step to ensure the consistency and integrity of glycoprotein samples, or to validate findings obtained through other glycan analysis techniques.

Integrate glycan and protein information to gain insights into glycoprotein structure, function, and potential implications in biological processes or disease. Bioinformatics tools and databases can aid in the analysis of this data.

Step 5 Functional Assays

Assess the functional significance of glycosylation on the glycoprotein by performing functional assays, such as binding studies, enzymatic activity assays, or cell-based assays, comparing glycosylated and deglycosylated forms.

Glycosylation can influence cell adhesion and interactions. Cell adhesion assays, such as cell aggregation or cell-substrate adhesion assays, can be performed to evaluate the impact of glycosylation on cellular behavior. By comparing the adhesion properties of glycosylated and deglycosylated glycoproteins, the significance of glycosylation in cell adhesion can be determined.

In some cases, glycosylation can directly affect the enzymatic activity of glycoproteins. Enzymatic activity assays can be employed to measure the catalytic function of glycosylated and deglycosylated forms of the protein. By comparing the enzyme activity between the two forms, the impact of glycosylation on enzymatic function can be assessed.

Glycosylation can play a crucial role in receptor-ligand interactions. Receptor-ligand binding assays, such as surface plasmon resonance (SPR) or lectin microarray, can be employed to evaluate the binding affinity and kinetics between glycosylated glycoproteins and their specific receptors. By comparing the binding properties with deglycosylated forms, the significance of glycosylation in receptor-ligand interactions can be determined.

Step 6 Share Your Results

Open sharing of data fosters collaboration and promotes scientific advancement. By making glycomics and glycoproteomics data available to the broader scientific community, researchers from diverse disciplines can leverage the information for their own studies, leading to new discoveries and insights.

Please visit out external resources page to learn about online reporting tools available for glycomics data analysis and reporting.

Upcoming Workshops

We're thrilled to announce that GIA will be hosting interactive workshops on how to incorporate glyco into your work. Check back for more information on these workshops, including dates, topics, and registration details at a later time. You can also follow us on Twitter to stay in the loop and catch the latest news as it unfolds.

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