Detecting Glycosylation

Glycosylation of protein is a widespread, structurally complex and varied post-translational modification of proteins, playing important roles in cells and organisms, such as participating in the regulation of cell recognition, adhesion, signal transduction, etc. The research process and content related to the glycosylation modification phenomenon of proteins are as follows: firstly, the identification of glycoproteins (glycosylated proteins), that is, determining whether there is glycosylation in the protein; secondly, the enrichment of glycoproteins, that is, the separation and purification of glycoproteins. Finally, the characterization of glycoproteins is divided into two aspects: one is the characterization of proteins in glycoproteins, that is, the identification of glycosylation sites; the other is the characterization of sugar chains in glycoproteins, that is, the determination of sugar composition and the measurement of sugar content. Here I will introduce how to distinguish whether protein has undergone glycosylation, that is, to detect the existence of glycosylation. The detection methods are as follows:

 

1. Radiolabeling Method

3 H or 14C labeled sugar is added to cultured cells or tissues, and glycoproteins are detected by radiographic autoradiography. The level of protein glycosylation can also be detected by radioactively labeling the receptors of glycoproteins, such as radioreceptor analysis.

 

Characteristic: It is mostly used in the research of virus-induced tumor cells. It has high detection sensitivity, but the process of radioactive labeling is slow and dangerous, and it is costly.

 

2. Molecular Fluorescence Method

Molecular fluorescence labeling method is a classic method for detecting glycoproteins. Since some glycoproteins have the property of autofluorescence, the level of protein glycosylation can be reflected by measuring the fluorescence value with a fluorescence spectrophotometer. Currently, the most widely used excitation wavelength is 370nm/emission wavelength 440nm.

 

Characteristic: It has good sensitivity and repeatability, but can only detect some glycoproteins and has poor specificity, and is easily affected by environmental factors.

 

3. Electrophoresis Method

Electrophoresis is a traditional method for detecting glycoproteins. The existence of glycoproteins can be detected by single or two-dimensional electrophoresis. The principle is that there is a difference in migration speed between glycoproteins and non-glycoproteins in the electric field. By comparing this difference, it can be inferred whether the protein has undergone glycosylation modification.

 

Characteristic: The operation is simple, and the result is straightforward. The disadvantage is that the resolution is limited, which may cause protein denaturation, and the electrophoresis process takes a long time.

 

4. Lectin Labeling Method

Currently the most commonly used method for detecting glycoproteins. Lectins are a type of protein that can recognize and bind to glycosyl structures. They usually have high specificity and affinity and can bind to specific glycosyl structures.

 

Characteristic: It has high specificity and sensitivity, can be used to detect different types of glycosylation modifications, but some lectins may have binding ability to multiple glycosyl structures, and cross-reactions may occur.

 

5. Antibody Labeling Method

The principle of antibody labeling method to measure glycosylation is based on the specific binding between antibody and antigen. This method requires the preparation of specific antibodies against specific sugar chain structures or glycosylation sites. These antibodies can specifically bind to the sugar chain structures on glycoproteins.

 

Characteristic: It has high sensitivity and specificity, but the preparation of antibodies is difficult, and the cost is also high.

 

6. Chemical Enzyme Method

By using specific enzymes to decompose the sugar and protein parts in glycosylated proteins, and by measuring the concentration of the decomposition products to indirectly determine the degree of protein glycosylation.

 

Characteristic: High accuracy, can specifically recognize glycosylation modifications, but this method may be limited by the detection of some sugar chain structures, and the operation is relatively complicated.