New method of peptide cleavage based on Edman degradation

Area of Science:

• Organic Chemistry
• Analytical Chemistry
• Biochemistry

Background:

• Solid-phase peptide synthesis (SPPS) is crucial for creating peptide libraries.
• Efficient cleavage and sequencing methods are essential for analyzing these libraries.
• Existing methods may have limitations in sensitivity or compatibility.

Purpose of the Study:

• To develop a straightforward and efficient method for cleaving N-acylated peptides from acid-stable resins.
• To enable highly sensitive peptide sequencing using ESI-MS/MS.
• To demonstrate the applicability of the method in combinatorial chemistry.

Main Methods:

• Design of a cleavable linker based on a lysine residue with orthogonal protecting groups.
• Peptide synthesis on TentaGel HL-NH resin.
• Cleavage via phenylthiohydantoin (PTH) formation using phenyl isothiocyanate and acidolysis.
• Application of a fixed charge tag (2-(4-aza-1-azoniabicyclo[2.2.2]octylammonium)acetyl) to enhance ionization.
• Analysis by Electrospray Ionization-tandem Mass Spectrometry (ESI-MS/MS).

Main Results:

• Successful cleavage of N-acylated peptides from acid-stable resins.
• Formation of stable PTH derivatives upon cleavage.
• Enhanced ionization efficiency and reduced detection limits due to the charge tag.
• Achieved ESI-MS/MS sequencing of peptides in the subfemtomolar range.
• Demonstrated compatibility with one-bead-one-compound peptide library synthesis.

Conclusions:

• The proposed cleavage strategy is efficient and compatible with solid-phase peptide synthesis.
• The use of a fixed charge tag significantly improves peptide detection limits for ESI-MS/MS.
• This method facilitates sensitive peptide sequencing and is applicable to combinatorial chemistry and peptide library generation.