Recombinant Protein Glycosylation Analysis

Recombinant Protein Glycosylation Analysis is a technique used for the detection and analysis of the glycan structure and composition of recombinant proteins. Glycosylation is a common post-translational modification of proteins, which significantly impacts the stability, activity, and cellular interactions of proteins. Recombinant Protein Glycosylation Analysis is particularly important for the biopharmaceutical industry because it ensures the quality and therapeutic efficacy of biopharmaceuticals. Some major glycosylation analysis techniques are as follows.

 

High-Performance Liquid Chromatography (HPLC)

1. HPLC after Glycosidase Digestion

The protein sample is treated with glycosidase to release the glycans, which are then analyzed by HPLC to identify the glycan composition and structure.

 

2. Fluorescence-Labeled Glycan Analysis

Glycans are labeled with specific fluorescent markers and then separated and detected by HPLC.

 

Mass Spectrometry (MS)

1. Matrix-Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry (MALDI-TOF MS)

This is used to analyze the type and degree of protein glycosylation and can provide information on the mass and composition of glycans.

 

2. Electrospray Ionization Mass Spectrometry (ESI-MS)

This can be used for the analysis of complex glycan structures, including the identification of glycosylation sites.

 

Nuclear Magnetic Resonance Spectroscopy (NMR)

NMR can provide detailed information on the structure of the glycan, including the way sugar residues are connected. NMR is very useful for determining the precise structure of complex glycans.

 

Capillary Electrophoresis (CE)

Capillary electrophoresis separates glycans by electrophoresis. This method is very effective for analyzing the heterogeneity of glycosylation and the charge state of the glycan.

 

Chip Technology

Glycan chips can rapidly screen and analyze many different sugar structures, making them suitable for high-throughput analysis.

 

Immunological Methods

Specific antibodies can be used to detect specific glycosylated structures, such as by methods like ELISA.

 

When performing Recombinant Protein Glycosylation Analysis, the protein sample first needs to be purified, then an appropriate analysis technique is chosen for the release, labeling, separation, and identification of the glycan. During the analysis, one must consider the type of glycosylation (N-glycosylation or O-glycosylation), glycan heterogeneity, and the site of glycosylation. The choice of glycosylation analysis technique depends on the purpose of the analysis, the complexity of the sample, and the required depth and accuracy of the analysis. When designing the experiment, one must also take into account the available amount of the sample, the required analysis time, and cost factors.