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本文依次采用Immobilized p-Aminophenyl Phosphoryl Choline Gel亲和层析柱和Superdex 75 10/300 GL分子筛层析柱分离纯化HEK293细胞重组C-反应蛋白(C-reactive protein,CRP),对其进行了凝胶电泳、酶切、MALDI-TOF-MS鉴定,并利用蛋白质酶解-同位素稀释质谱法对其进行准确定量。结果表明,重组得到的样品符合CRP结构特征,液相纯度为96.527%,蛋白质酶解-同位素稀释质谱法定量的结果为3.32mg/g,扩展不确定度为0.19mg/g(k=2)。本文建立的蛋白质酶解-同位素稀释质谱法不受样品中肽类/蛋白类杂质的影响,提高了C-反应蛋白含量测定的准确度和精密度,定量结果可溯源至SI单位,为C-反应蛋白标准物质的研制奠定了基础。
In this paper, recombinant C-reactive protein (CRP) of HEK293 cells was isolated and purified by using Immobilized p-Aminophenyl Phosphoryl Choline Gel affinity column and Superdex 75 10/300 GL molecular sieve chromatography column, Electrophoresis, enzyme digestion, MALDI-TOF-MS identification, and the use of protein hydrolysis - isotope dilution mass spectrometry accurate quantification. The results showed that the reconstructed samples were in accordance with the structural features of CRP, the liquid phase purity was 96.527%, the protein enzymolysis-isotope dilution mass spectrometry was 3.32mg / g, the extended uncertainty was 0.19mg / g (k = 2) . The proteolysis-isotope dilution mass spectrometry established in this paper is not affected by peptide / protein impurities in the sample and improves the accuracy and precision of the determination of C-reactive protein content. Quantitative results can be traced back to SI units for C- Preparation of reference protein reagents laid the foundation.