Principle of Peptide Structure Determination

Peptides are linear molecules composed of amino acids linked by peptide bonds, playing vital roles in numerous biological functions. Determining the structure of peptides is crucial for understanding their functions, as the primary structure (amino acid sequence), secondary structure (local folding), tertiary structure (overall three-dimensional conformation), and quaternary structure (complexes of multiple peptide chains) significantly influence their biological activity. Peptide structure determination primarily involves the identification of the amino acid sequence, secondary structure prediction, tertiary structure elucidation, and identification of chemical modification sites.

 

Amino Acid Sequence Determination

The primary structure of a peptide refers to the specific order of amino acids, and determining this sequence is the first step in peptide structure analysis. Common methods for peptide sequencing include Edman degradation, mass spectrometry (MS), and nuclear magnetic resonance (NMR) spectroscopy.

 

1. Edman Degradation

Edman degradation is a classical chemical degradation method, which sequentially cleaves and identifies the N-terminal amino acids of a peptide. In this method, the peptide reacts with phenyl isothiocyanate (PITC) to form a PITC-amino acid derivative. This derivative is then cleaved under acidic conditions, releasing the N-terminal amino acid as a stable acyl derivative. Chromatography is subsequently used to separate and identify this derivative, revealing the first amino acid in the sequence. This process can be repeated to sequentially determine the peptide's full sequence. However, Edman degradation is less efficient for longer peptides and is typically used for short peptides or peptide fragments.

 

2. Mass Spectrometry (MS)

Mass spectrometry is an analytical method based on molecular mass, enabling efficient and accurate peptide sequencing. Electrospray ionization mass spectrometry (ESI-MS) or matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is commonly used to ionize peptide molecules and determine their molecular weights. Using tandem mass spectrometry (MS/MS), peptides are fragmented stepwise, and the differences in mass between fragments allow the deduction of the amino acid sequence. MS is capable of handling longer peptides and offers high throughput and sensitivity, making it widely employed in proteomics research.

 

3. Nuclear Magnetic Resonance (NMR) Spectroscopy

Nuclear magnetic resonance (NMR) spectroscopy determines molecular structures by detecting the resonance frequencies of atomic nuclei in an external magnetic field. In the context of primary structure determination, NMR is often used to confirm specific amino acid residues in a peptide chain. Although NMR has lower resolution and is more limited in application for large peptides, it is invaluable for studying small peptides and their interactions with other molecules.

 

Additional Techniques for Peptide Sequencing

In addition to the methods mentioned above, enzymatic digestion combined with mass spectrometry is also a common strategy for peptide structure determination. Specific proteases, such as trypsin, cleave peptides into smaller fragments, which can then be analyzed using mass spectrometry to reconstruct the full peptide sequence. Additionally, X-ray crystallography and cryo-electron microscopy (Cryo-EM) can be used to elucidate the three-dimensional structure of peptides, aiding in understanding the relationship between sequence and function.

 

Peptide structure determination extends beyond primary structure to include secondary and tertiary structures. Techniques such as circular dichroism (CD) and infrared spectroscopy (IR) are commonly used for secondary structure prediction, while X-ray crystallography, NMR, and cryo-electron microscopy are primarily employed for tertiary structure determination. The identification of modification sites (e.g., phosphorylation, glycosylation) is also an essential aspect of peptide structure determination, achieved through modification-specific mass spectrometry, providing insights into peptide bioactivity and regulatory mechanisms.