A selective fluorescence reaction for peptides and chromatographic analysis

Area of Science:

• Analytical Chemistry
• Biochemistry

Background:

• Accurate peptide quantification is crucial for various biological and chemical analyses.
• Existing methods may lack sensitivity or selectivity for complex peptide mixtures.

Purpose of the Study:

• To develop a novel and selective fluorescence reaction for the quantitative determination of peptides.
• To establish a method compatible with reversed-phase liquid chromatography (RPLC).

Main Methods:

• A new fluorescence reaction involving catechol, sodium periodate, and sodium borate at neutral pH was employed.
• Peptides were heated to form a single fluorescent product.
• Separation was achieved using RPLC with gradient elution of methanol.
• Quantitative detection was performed using fluorimetry.
• Product structures were elucidated using electrospray ionization-time of flight-mass spectrometry (ESI-TOF/MS).

Main Results:

• A single fluorescent product was consistently formed from peptides under the specified reaction conditions.
• Four peptides (Leu-Gly, Ala-Leu-Gly, Tyr-Gly-Gly-Phe-Leu, Leu-Leu-Leu) were successfully separated and quantified.
• The method demonstrated high sensitivity with lower detection limits of 0.5-1.0 pmol.
• The chemical structures of fluorescent products from phenylalanine amide and Leu-Leu-Leu were identified.

Conclusions:

• The proposed fluorescence reaction is a selective and sensitive method for peptide quantification.
• The developed RPLC-based method offers efficient separation and detection of peptides.
• This technique provides a valuable tool for biochemical analysis and peptide research.