Quantitative 1H Nuclear Magnetic Resonance (qNMR) of Aromatic Amino Acids for Protein Quantification

Abstract

Hydrolysis of protein samples into amino acids facilitates the use of NMR spectroscopy for protein and peptide quantification. Different conditions have been tested for quantifying aromatic amino acids and proteins. The pH-dependent signal shifts in the aromatic region of amino acid samples were examined. A pH of 12 was found to minimize signal overlap of the four aromatic amino acids. Several aromatic compounds, such as terephthalic acid, sulfoisophthalic acid, and benzene tricarboxylic acid, were applied as internal standards. The quantification of amino acids from an amino acid standard was performed. Using the first two suggested internal standards, recovery was ~97% for histidine, phenylalanine, and tyrosine at a concentration of approximately 1 mM in solution. Acidic hydrolysis of a certified reference material (CRM) of bovine serum albumin (BSA) and subsequent quantification of Phe and Tyr yielded recoveries of 98% ± 2% and 88% ± 4%, respectively, at a protein concentration of 16 g/L or 250 µM.

Simple Determination of Affinity Constants of Antibodies by Competitive Immunoassays

“…Since the concentration of the antigen is crucial for the accuracy of the affinity constant, as it is directly reflected in deviations from the "true" affinity constant, the impurities should be taken into account if a high accuracy of the result is important. The most common method for a reliable determination of the peptide concentration seems to be amino acid analysis, e.g., [44][45][46][47], which is sometimes offered by companies performing custom peptide synthesis. If an identical calibration substance of known purity is available, many other methods might be suitable [48], such as HPLC with UV detection.…”

Simple Determination of Affinity Constants of Antibodies by Competitive Immunoassays

“…Since the concentration of the antigen is crucial for the accuracy of the affinity constant, as it is directly reflected in deviations from the “true” affinity constant, the impurities should be taken into account if a high accuracy of the result is important. The most common method for a reliable determination of the peptide concentration seems to be amino acid analysis, e.g., [ 57 , 58 , 59 , 60 ], which is sometimes offered by companies performing custom peptide synthesis. If an identical calibration substance of known purity is available, many other methods might be suitable [ 61 ], such as HPLC with UV detection.…”