Table of Contents
Journal of Amino Acids
Volume 2016 (2016), Article ID 7374316, 8 pages
http://dx.doi.org/10.1155/2016/7374316
Research Article

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

Division 1.5 Protein Analysis, Bundesanstalt für Materialforschung und -prüfung (BAM), Richard-Willstätter-Strasse 11, 12489 Berlin, Germany

Received 15 April 2016; Accepted 28 June 2016

Academic Editor: Hieronim Jakubowski

Copyright © 2016 Almut Hesse and Michael G. Weller. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Amino acid analysis is considered to be the gold standard for quantitative peptide and protein analysis. Here, we would like to propose a simple HPLC/UV method based on a reversed-phase separation of the aromatic amino acids tyrosine (Tyr), phenylalanine (Phe), and optionally tryptophan (Trp) without any derivatization. The hydrolysis of the proteins and peptides was performed by an accelerated microwave technique, which needs only 30 minutes. Two internal standard compounds, homotyrosine (HTyr) and 4-fluorophenylalanine (FPhe) were used for calibration. The limit of detection (LOD) was estimated to be 0.05 µM (~10 µg/L) for tyrosine and phenylalanine at 215 nm. The LOD for a protein determination was calculated to be below 16 mg/L (~300 ng BSA absolute). Aromatic amino acid analysis (AAAA) offers excellent accuracy and a precision of about 5% relative standard deviation, including the hydrolysis step. The method was validated with certified reference materials (CRM) of amino acids and of a pure protein (bovine serum albumin, BSA). AAAA can be used for the quantification of aromatic amino acids, isolated peptides or proteins, complex peptide or protein samples, such as serum or milk powder, and peptides or proteins immobilized on solid supports.