Protein Primary Structure Determination: Decoding Linear Amino Acid Sequences

Determining the primary structure of a protein, that is, determining the linear sequence of amino acids, is one of the foundations of molecular biology research. Since the first determination of the amino acid sequence of insulin in the 1950s, the technology has made great progress. Here are the basic steps and some common methods for determining the primary structure of a protein:

 

Protein Purification

First, the target protein needs to be separated from other proteins and impurities.

 

Cutting Protein

Proteins may be very long, and directly determining their amino acid sequence may be difficult. Therefore, specific proteases (such as trypsin or pepsin) are typically used to cut the protein into shorter peptide segments.

 

Peptide Sequence Sorting

Through multiple cuts and different enzymes, overlapping peptide segments can be obtained, which can help arrange the entire amino acid sequence of the protein.

 

Determining the Amino Acid Sequence of the Peptide

1. Edman Degradation

This is a classic technique that determines the sequence by gradually removing the N-terminal amino acids of the peptide chain. Although this process is relatively slow, it is very accurate and particularly suitable for shorter peptide segments.

 

2. Mass Spectrometry

Modern techniques tend to use mass spectrometry to determine the amino acid sequence of proteins and peptide segments. Through mass spectrometry, the mass of molecules can be measured, and the order of amino acids can be determined based on mass.

 

Reconstructing the Protein Sequence

Once all the peptide segments have been sequenced, the entire amino acid sequence of the protein can be reconstructed by finding the overlapping regions between these peptide segments.

 

Comparing with a Database

The obtained amino acid sequence can be compared with known protein sequence databases to determine whether it is similar or identical to other known proteins.

 

With the advancement of technology, especially mass spectrometry, the determination of the primary structure of proteins has become faster and more accurate. In addition, as more proteins are sequenced and stored in public databases, researchers can more easily identify and compare newly discovered proteins.