Approaches towards the quantitative analysis of peptides and proteins by reversed-phase high-performance liquid chromatography in the absence of a pure reference sample

A reversed-phase HPLC protocol for the quantitative analysis of peptides and proteins is presented. It is applicable to purified samples and potentially to crude biological extracts. The key feature is that an analytically pure reference sample of the analyte is not required because the extinction coefficient for the UV absorbance at 280 nm can be accurately estimated from the amino acid sequence. The concentration of a protein can therefore be calculated from the peak area relative to an internal standard. Sources of error and limitations of the method are systematically considered. Tryptophan containing peptides gave closer agreement to expected values than those with only tyrosine. It was found that analogous, previously used methods could not be directly applied to lower wavelengths.