Stepwise sequence determination from the carboxyl terminus of peptides

Area of Science:

• Biochemistry
• Analytical Chemistry
• Proteomics

Background:

• The thiocyanate method offers a route for stepwise peptide degradation from the carboxyl (COOH) terminus.
• Investigating and optimizing this established method is crucial for accurate peptide analysis.

Purpose of the Study:

• To investigate and optimize the thiocyanate method for stepwise peptide degradation.
• To develop efficient conditions for removing and identifying COOH-terminal residues.
• To adapt the method for solid-phase peptide analysis.

Main Methods:

• Utilized model peptides to study the two-step degradation process (thiocyanate activation and acetohydroxamate cleavage).
• Developed a solid-phase version using porous glass supports functionalized with N,N'-carbonyldiimidazole.
• Employed high-pressure liquid chromatography (HPLC) and gas-liquid chromatography (GLC) for 2-thiohydantoin identification.

Main Results:

• Established conditions for rapid and efficient removal and identification of COOH-terminal residues.
• Successfully applied the solid-phase degradation to immobilized peptides over multiple rounds.
• Demonstrated the method's utility in sequencing short peptides and determining the COOH-terminal sequence of a myoglobin fragment.

Conclusions:

• The optimized thiocyanate method, particularly in its solid-phase format, provides a robust tool for peptide sequencing.
• HPLC and GLC offer sensitive and reliable methods for identifying cleaved amino acid derivatives.
• This approach facilitates the analysis of peptide sequences, including those from larger protein fragments.