Center for Bioanalysis, Department of Chemical and Medical Metrology, Korea Research Institute of Standards and Science, 267 Gajeong-ro, Youseong-gu, Daejeon 34113, Republic of Korea; Department of Bio-Analytical Science, University of Science and Technology, 217 Gajeong-ro, Yuseong-gu, Daejeon 34113, Republic of Korea.
Center for Bioanalysis, Department of Chemical and Medical Metrology, Korea Research Institute of Standards and Science, 267 Gajeong-ro, Youseong-gu, Daejeon 34113, Republic of Korea; College of Pharmacy, Kyung Hee University, Seoul 02447, Republic of Korea.
J Chromatogr B Analyt Technol Biomed Life Sci. 2019 Sep 15;1126-1127:121732. doi: 10.1016/j.jchromb.2019.121732. Epub 2019 Jul 25.
A certified reference material (CRM) for the quantification of protein, essential to manage quality control and quality assurance in protein-related works, has been developed. Amino acid analysis with conventional acid hydrolysis and isotope dilution HPLC-MS was used to establish an SI-traceable absolute protein quantification method using recombinant human growth hormone (hGH) as a model protein. The certification method was verified by comparative studies between 1) different methods of protein quantification based on microwave-assisted hydrolysis, and 2) different labs as part of the Asian Collaboration on Reference Material project with Japan, China, and Korea. Certification, evaluation of measurement uncertainty, homogeneity testing, and stability testing were carried out, after which the candidate CRM for hGH quantification was successfully certified with excellent agreement within the certified value in the two comparative studies. Although the quantification value of hGH by amino acid analysis showed good robustness in various conditions, results of intact protein analysis showed degradation profiles in temperatures higher than 4 °C. Consequently, storage and dissemination conditions should be set in accordance with stability tests. Based on the results, this method is believed to be suitable for accurate quantification of hGH. Additionally, it can also be used as a guide to preparation of CRM, and instructions for quality management of protein work for other similar proteins.
已开发出一种用于蛋白质定量的认证参考物质（CRM），这对于蛋白质相关工作的质量控制和质量保证至关重要。采用常规酸水解和同位素稀释 HPLC-MS 氨基酸分析，建立了一种 SI 可溯源的绝对蛋白质定量方法，以重组人生长激素（hGH）为模型蛋白。通过对 1）基于微波辅助水解的不同蛋白质定量方法，和 2）日本、中国和韩国的亚洲参考物质合作项目中的不同实验室之间的比较研究，对认证方法进行了验证。进行了认证、测量不确定度评估、均匀性检验和稳定性检验，之后候选 hGH 定量 CRM 在两个比较研究中成功地进行了认证，在认证值范围内具有优异的一致性。尽管氨基酸分析的 hGH 定量值在各种条件下表现出良好的稳健性，但完整蛋白质分析的结果表明，在高于 4°C 的温度下会出现降解谱。因此，应根据稳定性试验设定储存和传播条件。基于这些结果，该方法被认为适用于 hGH 的准确定量。此外，它还可以用作 CRM 制备的指南，并为其他类似蛋白质的蛋白质工作质量管理提供指导。
