Moffatt F, Senkans P, Ricketts D
Zeneca Agrochemicals, Jealotts Hill Research Station, Bracknell, Berks, UK.
J Chromatogr A. 2000 Sep 8;891(2):235-42. doi: 10.1016/s0021-9673(00)00620-8.
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.
本文介绍了一种用于肽和蛋白质定量分析的反相高效液相色谱方法。该方法适用于纯化样品，也可能适用于粗生物提取物。其关键特性在于，由于可以根据氨基酸序列准确估算280 nm处紫外吸光度的消光系数，因此无需分析纯的被分析物参考样品。因此，可以根据相对于内标物的峰面积计算蛋白质的浓度。系统地考虑了该方法的误差来源和局限性。含色氨酸的肽比仅含酪氨酸的肽与预期值的一致性更高。研究发现，以前使用的类似方法不能直接应用于更低波长。
