Detection Methods for Changes in Protein Glycosylation Levels

Glycosylation is a crucial post-transcriptional modification process that influences the folding, stability, activity, and intercellular interactions of proteins.

 

There are various methods to determine if the glycosylation modification level on a protein has changed, and each method has its characteristics and applicable scenarios. Here are some commonly used techniques:

 

1. Mass Spectrometry (MS)

MS is a very powerful tool, especially when combined with liquid chromatography (LC-MS/MS), to identify detailed information about the glycosylation site, type, and degree of modification on a protein. The protein is first enzymatically digested into peptides, and then the mass and sequence of the glycopeptides are analyzed by mass spectrometry. By comparing the relative abundance of the same glycopeptides in different samples, changes in the level of glycosylation can be quantitatively analyzed.

 

2. Western Blot

Using specific antibodies, the level of glycosylation of specific proteins can be detected. Glycosylation modification usually affects the molecular weight of the protein, so changes in migration rate can be observed in gel electrophoresis.

 

3. Lectin Affinity Chromatography

Lectins specifically bind to glycosylated proteins or peptides. Specific types of glycosylated proteins can be enriched through lectin affinity chromatography and then further analyzed, such as by mass spectrometry.

 

4. Flow Cytometry

If the target protein is expressed on the cell surface, flow cytometry can be used in combination with glycosylation-specific antibodies to detect the glycosylation state of cell surface proteins.

 

5. HPLC (High Performance Liquid Chromatography)

HPLC can be used to analyze the released glycan chains of proteins. Different types of glycan chains are separated by different types of chromatography columns (such as hydrophilic interaction chromatography, reverse phase chromatography, etc.). Combined with fluorimetric or mass spectrometric detectors, quantitative and qualitative analysis of glycan chains can be achieved.

 

6. ELISA (Enzyme-Linked Immunosorbent Assay)

This is a commonly used quantitative analysis method that uses specific antibodies to detect changes in protein glycosylation levels.

 

Each method has its scope and limitations. For example, mass spectrometry provides the most detailed information, but the equipment cost is high, and the technical requirements are strict; Western blot is relatively simple, but may not be as precise as mass spectrometry. Therefore, the choice of method depends on the specific purpose of the experiment and available resources.