Exploring Cutting-Edge Techniques in Protein Sequencing: From Basics to Applications

Determining the amino acid sequence of proteins is fundamental to understanding their functions, structures, and interactions. This process involves a series of techniques, each with its unique advantages and limitations. Here are some commonly used protein sequencing methods.

 

Mass Spectrometry

Proteins or peptides are broken down into smaller fragments, and then a mass spectrometer is used to measure the mass-to-charge ratio of these fragments, thereby inferring the amino acid sequence. This is typically achieved through fragmentation techniques such as collision-induced dissociation or electron transfer dissociation.

 

1. Advantages

It has high sensitivity and it's capable of analyzing complex samples and mixtures, suitable for large-scale proteomics studies.

 

2. Disadvantages

High equipment cost, requiring specialized operation and data analysis skills.

 

Edman Degradation

This is a classic chemical method, which removes the amino acids at the N-terminus of the protein one by one, and identifies the released amino acid at each step.

 

1. Advantages

Mature method, non-destructive to other parts of the protein.

 

2. Limitations

The analysis process is slow, usually only suitable for shorter protein sequences (50 amino acid residues or less).

 

X-Ray Crystallography

The precise structure of a protein in three-dimensional space is determined by measuring the scattering of X-rays when passing through a protein crystal, thereby inferring the amino acid sequence.

 

1. Advantages

It can provide detailed information about the three-dimensional structure of the protein.

 

2. Limitations

It requires high-quality protein crystals, and the experimental process is complex and time-consuming.

 

Nuclear Magnetic Resonance Spectroscopy

It uses magnetic resonance technology to determine the atomic-level structure and dynamic information of the protein.

 

1. Advantages

It does not require a crystalline form of the sample and can study proteins in solution.

 

2. Limitations

It's usually limited to proteins with a smaller molecular weight.

 

Gene-Based Protein Sequencing

The gene corresponding to the protein is sequenced, and then bioinformatics tools are used to translate the DNA sequence into an amino acid sequence.

 

1. Advantages

It's suitable for proteins whose sequences cannot be obtained by direct protein sequencing methods.

 

2. Limitations

It can not detect any post-translational modifications of the protein.