Structure analysis of peptides

Primary structure analysis of peptides refers to determining the sequence of amino acids in a peptide chain. This sequence is fundamental because it dictates the peptide's higher-order structures and functions. Here’s a step-by-step guide to performing primary structure analysis, along with examples:

1. Amino Acid Composition Analysis

• Purpose: To determine the types and quantities of amino acids in the peptide.
• Method:
  • Acid Hydrolysis: The peptide is hydrolyzed in strong acid (like 6 M HCl) at high temperatures (110°C) for 24 hours. This breaks the peptide bonds, releasing free amino acids.
  • Chromatography: The amino acids are then separated and quantified using techniques like High-Performance Liquid Chromatography (HPLC).
• Example: Suppose the analysis reveals that the peptide consists of alanine (Ala), glycine (Gly), and serine (Ser). This tells us the composition but not the sequence.

2. Edman Degradation

• Purpose: To sequentially determine the amino acid sequence from the N-terminus of the peptide.
• Method:
  • Phenylisothiocyanate (PITC) reacts with the N-terminal amino acid to form a phenylthiocarbamoyl derivative.
  • Acid Treatment: This derivative is then cleaved from the peptide, forming a stable phenylthiohydantoin (PTH)-amino acid, which can be identified using chromatography.
  • Repetition: The process is repeated for each subsequent amino acid in the sequence.
• Example: If the peptide sequence is Ala-Gly-Ser, Edman degradation would sequentially release and identify Ala first, followed by Gly, then Ser.

3. Mass Spectrometry (MS)

• Purpose: To determine the molecular mass and sequence of peptides.
• Method:
  • Electrospray Ionization (ESI) or Matrix-Assisted Laser Desorption/Ionization (MALDI) are common ionization techniques used.
  • Tandem Mass Spectrometry (MS/MS): After ionization, peptides are fragmented in the mass spectrometer, and the masses of the fragments are measured. The differences between the masses of successive fragments correspond to the mass of an amino acid residue, allowing for sequence determination.
• Example: A peptide with the sequence Ala-Gly-Ser might show peaks corresponding to the mass differences between Ala-Gly, Gly-Ser, etc., allowing the sequence to be deduced.

4. Peptide Mapping

• Purpose: To identify the sequence by comparing it with known sequences.
• Method:
  • The peptide is enzymatically or chemically cleaved into smaller fragments.
  • These fragments are analyzed by HPLC or MS and compared to a database of known peptide sequences.
• Example: If the peptide is known to be part of a larger protein, peptide mapping can confirm its exact position within that protein by matching the fragments to the expected sequence.

5. Automated Sequencing

• Purpose: To obtain the complete sequence of a peptide.
• Method: Instruments like the sequenator automate the Edman degradation process, providing rapid and accurate sequence information.
• Example: A sequenator could quickly determine the sequence of a peptide like Ala-Gly-Ser-Thr-Lys.

6. Bioinformatics Tools

• Purpose: To predict or confirm peptide sequences using computational methods.
• Method:
  • Sequence Alignment: Tools like BLAST (Basic Local Alignment Search Tool) can be used to compare the peptide sequence against databases to find similar sequences.
• Example: If a peptide sequence Ala-Gly-Ser-Thr-Lys is input into a database, it may match with a known peptide or protein, helping confirm the sequence.

Summary Example:

Imagine you are analyzing a peptide suspected to be Ala-Gly-Ser-Thr-Lys. Here's how you might approach it:

1. Hydrolysis and HPLC confirm the presence of Ala, Gly, Ser, Thr, and Lys.
2. Edman Degradation sequentially identifies Ala as the N-terminal amino acid, followed by Gly, Ser, Thr, and Lys.
3. Mass Spectrometry confirms the sequence by analyzing the mass of the entire peptide and its fragments.
4. Peptide Mapping might compare the sequence to a known protein to confirm its identity.
5. Automated Sequencing could quickly verify the sequence without manual steps.

Conclusion

Primary structure analysis is a crucial step in understanding a peptide's function and properties. Combining methods like Edman degradation, mass spectrometry, and bioinformatics tools provides a comprehensive approach to accurately determining the amino acid sequence of peptides.