Amino Acid Sequence Determination Methods

Amino acid sequence determination, also known as protein sequence determination, refers to the process of determining the exact order of amino acid residues in a protein. This information is vital for understanding the structure and function of proteins, identifying new proteins, studying protein interactions, and disease associations. Here are some of the main methods for amino acid sequence determination:

 

1. Edman Degradation

This is a classic method for protein sequence analysis, involving the stepwise, selective removal of the N-terminal (end) amino acid of a protein or peptide for identification. This process can be repeated multiple times to determine the amino acid sequence one by one.Although this method is very effective, it is typically limited to shorter polypeptide chains, as reduced reaction efficiency on longer chains can impact the accuracy of the results.

 

2. Mass Spectrometry

Mass spectrometry is a powerful analytical technique for determining the mass or mass-to-charge ratio (m/z) of amino acids, from which the amino acid sequence can be inferred.In protein mass spectrometry, proteins or peptides are usually first cut into smaller fragments by an enzyme (like trypsin), and these fragments are then introduced into the mass spectrometer for analysis.Tandem mass spectrometry (MS/MS) can provide further detailed information about peptide segments, aiding accurate determination of the amino acid sequence.

 

3. Protein/Nucleic Acid Database Comparison

If the gene of the protein to be sequenced is known, the amino acid sequence can be predicted by translating the nucleotide sequence of that gene. This involves the use of bioinformatics tools and databases, like NCBI's BLAST, etc. Additionally, the known amino acid sequence of a protein can be compared with sequences in a database to discover possible homologies and function-relatedness.

 

Each method has its specific advantages and limitations, so researchers may choose an appropriate strategy based on specific experimental conditions and needs. With the advancement of technology, new sequencing technologies and instruments continue to emerge, aiding faster and more accurate determination of amino acid sequences.