How do you find the amino acid sequence in a protein?

There are two primary methods used to determine the amino acid sequence of a protein: mass spectrometry and Edman degradation.

Mass Spectrometry

• Most Common Method: Mass spectrometry is currently the most widely used method due to its ease of use and efficiency.
• Principle: It measures the mass-to-charge ratio of ions. In the context of proteins, this is achieved by breaking down the protein into smaller peptides. The resulting peptides are then analyzed and used to infer the original amino acid sequence.
• Process:
  1. Protein Digestion: The protein is typically digested into smaller peptides using enzymes (like trypsin).
  2. Ionization: The peptides are then ionized.
  3. Mass Analysis: The mass-to-charge ratio of these ions is measured, providing information about their masses.
  4. Sequence Determination: Algorithms and software are employed to piece together the amino acid sequence from the measured peptide masses.
• Advantages: High throughput, sensitive, and requires relatively small amounts of sample.

Edman Degradation

• Historical Method: This method is older but still used, especially for characterizing the N-terminus of a protein.
• Protein Sequenator: This process is done using a machine called a protein sequenator.
• Principle: It sequentially removes one amino acid at a time from the N-terminus of the protein.
• Process:
  1. Coupling: A chemical reagent, phenylisothiocyanate (PITC), is added to the protein, which reacts with the N-terminal amino acid.
  2. Cleavage: Under acidic conditions, the N-terminal amino acid derivative is cleaved off, leaving the rest of the protein chain intact.
  3. Identification: The cleaved amino acid derivative is then analyzed using chromatography.
  4. Repetition: The process is repeated multiple times, each time removing and identifying the next N-terminal amino acid until the protein sequence is determined.
• Advantages: Precise identification of the N-terminal amino acid, good for smaller peptides.
• Limitations: Not as high throughput as mass spectrometry and may have issues with long protein sequences.

Summary Table

Method	Description	Advantages	Limitations
Mass Spectrometry	Measures mass-to-charge ratio of ionized peptide fragments to infer protein sequence.	High throughput, sensitive, less sample needed.	May require complex data analysis, can be challenging with large proteins
Edman Degradation	Sequentially removes and identifies one amino acid at a time from the N-terminus of the protein.	Precise N-terminal identification, good for smaller sequences.	Lower throughput, less effective for large proteins.

Both methods contribute to our understanding of protein structure. The choice between the two often depends on factors such as the size and complexity of the protein being analyzed, as well as specific research goals.