Protein Quantification by Derivatization-Free High-Performance Liquid Chromatography of Aromatic Amino Acids

Area of Science:

• Analytical Chemistry
• Biochemistry

Background:

• Amino acid analysis is crucial for peptide and protein quantification.
• Existing methods can be time-consuming or require derivatization.

Purpose of the Study:

• To develop a simple, rapid, and accurate HPLC/UV method for aromatic amino acid analysis.
• To quantify tyrosine, phenylalanine, and tryptophan without derivatization.

Main Methods:

• Reversed-phase HPLC/UV separation of aromatic amino acids.
• Accelerated microwave-assisted hydrolysis (30 minutes).
• Use of homotyrosine and 4-fluorophenylalanine as internal standards.

Main Results:

• Achieved a limit of detection of 0.05 µM for tyrosine and phenylalanine.
• Protein determination limit below 16 mg/L.
• Demonstrated accuracy and precision of ~5% relative standard deviation, including hydrolysis.

Conclusions:

• The developed aromatic amino acid analysis (AAAA) method is accurate and precise.
• Applicable to diverse samples including complex biological matrices and immobilized proteins.
• Offers a rapid and efficient alternative for quantitative peptide and protein analysis.