The Principle of Peptide Sequencing

Peptide sequencing involves determining the primary structure of a protein by analyzing the amino acid sequence of peptide fragments. Understanding the amino acid sequence of proteins is crucial for studying their function, structure, and interactions. The development of peptide sequencing technology has advanced proteomics research, enabling scientists to gain deeper insights into the mechanisms of biomolecules.

 

The fundamental principle of peptide sequencing is to determine the order of amino acid residues in a peptide chain sequentially using chemical or physical methods. Peptide fragments are typically obtained from proteins through protease digestion or chemical cleavage. The sequence of the peptide fragments is then determined using techniques such as mass spectrometry or Edman degradation.

 

Mass Spectrometry

Mass spectrometry (MS) is one of the most widely used methods for peptide sequencing. The basic principle involves ionizing and separating peptide fragments based on their mass-to-charge ratio (m/z). The main steps in mass spectrometry are:

 

1. Ionization

Peptide fragments in the sample are ionized using methods such as electrospray ionization (ESI) or matrix-assisted laser desorption/ionization (MALDI). Electrospray ionization is suitable for liquid chromatography-mass spectrometry (LC-MS), while matrix-assisted laser desorption/ionization is commonly used in matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS).

 

2. Mass Analysis

The ionized peptide fragments are introduced into a mass spectrometer, where they are separated according to their mass-to-charge ratio by a mass analyzer, such as a quadrupole, time-of-flight analyzer, or Fourier transform ion cyclotron resonance analyzer.

 

3. Data Interpretation

The mass spectrum provides information about the mass-to-charge ratio of the peptide fragments. The amino acid sequence of the peptide fragments can be inferred by comparing with known peptide mass data in databases or by further fragmenting the peptides using tandem mass spectrometry (MS/MS) and analyzing the fragment ions.

 

Edman Degradation

Edman degradation is a classic chemical sequencing method suitable for determining the sequence of short peptides. The basic principle involves selectively cleaving and identifying amino acids from the N-terminus of the peptide chain step-by-step. The main steps in Edman degradation are:

 

1. Sample Preparation

The peptide fragments are immobilized on a solid-phase support to ensure efficient reaction.

 

2. Chemical Reaction

Phenylisothiocyanate (PITC) reacts with the N-terminal amino acid of the peptide to form a PTH-amino acid. The PTH-amino acid is then cleaved from the peptide chain through acid hydrolysis and released.

 

3. Analysis and Detection

The released PTH-amino acid is separated and detected using high-performance liquid chromatography (HPLC). The amino acid type is determined by comparing the retention time with standards.

 

Peptide sequencing is a crucial technology widely used in proteomics, disease research, and biomedicine. Mass spectrometry and Edman degradation each have their advantages and disadvantages. Mass spectrometry is suitable for large-scale, high-throughput peptide sequencing, while Edman degradation is ideal for precise, small-scale sequencing. With ongoing advancements, peptide sequencing methods will become more efficient and accurate, providing more powerful tools for life science research.