N-Sugar Spectrum Analysis

N-glycan profiling is a technique used to analyze the structure and composition of protein N-linked glycans. These glycans are a common type of biomolecule widely present in eukaryotes, especially on the cell surface and in secreted proteins. N-glycan profiling is crucial for understanding protein function, cell signaling, cell recognition, and disease mechanisms.

 

Analysis Workflow

1. Sample Preparation

Protein samples are extracted from the target protein or complex. This may involve purification and concentration steps to ensure the accuracy and efficiency of glycan profiling.

 

2. Release of N-Glycans

Protein samples are treated with specific enzymes, such as peptide-N-glycosidase F (PNGase F), to release N-linked glycans. This step is necessary because the glycans are covalently attached to the protein.

 

3. Glycan Purification

The released N-glycans may need to be further purified by chromatography or other methods to remove proteins, peptides, and other substances that may interfere with the analysis.

 

4. Labeling (Optional)

To improve detection sensitivity and resolution, the purified N-glycans can be labeled with fluorescence or other types of labels.

 

5. Separation and Detection

N-glycans are separated and detected using techniques such as high-performance liquid chromatography (HPLC), capillary electrophoresis (CE), or mass spectrometry (MS). These technologies can provide qualitative and quantitative information about the glycans.

 

6. Glycan Structure Identification

Detailed identification of the glycan structure is performed by further mass spectrometric analysis of the separated glycans, in combination with enzymatic and chemical methods. This may involve fragmentation analysis of the glycans to determine the sequence and linkage of the sugar units.

 

7. Data Analysis

Specialized software and databases are used to analyze and interpret the obtained data to determine the composition, structure, and diversity of the glycans. This includes comparing glycan profiles under different samples or conditions.

 

Applications

N-glycan profiling is vital for understanding protein function, stability, and intermolecular interactions, especially in the fields of biopharmaceuticals (such as the production of therapeutic antibodies) and disease diagnosis.

 

1. Biomedical Research

N-glycan profiling is critical in the development and quality control of biopharmaceuticals, such as antibody drugs.

 

2. Disease Diagnosis and Research

Certain diseases can affect the glycan patterns on the cell surface, so this technique plays a role in the identification of disease markers and mechanism research.

 

3. Cell Biology and Immunology

Analyzing changes in the glycans on the cell surface helps to understand cell interactions and signal transduction.