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目的:建立基于特征肽段的新冠病毒刺突蛋白S1亚基定量方法。方法:根据蛋白序列、糖基化修饰位点等筛选特征肽段;利用胰蛋白酶水解蛋白为相对分子质量较小的肽段,并优化反应参数;最后结合同位素稀释质谱法建立刺突蛋白S1亚基定量检测方法,并进行方法学考察。结果:基于特征肽段的同位素稀释质谱法检测样品中S1亚基质量分数为1.007×10-3,扩展不确定度为0.070×10-3,结果准确,与基于氨基酸分析的同位素稀释质谱法检测结果比较无显著差异。特征肽段质量分数在0.47×10-9～1×10-3范围内,回归相关系数R2>0.999 9,线性良好。该方法日内与日间精密度均小于5%,检测准确度均在95%～105%之间,重复性良好。该方法不受基质中其他蛋白的干扰,可溯源至国际单位制,有助于该蛋白绝对定量、标准物质研制等。结论:该方法可行性良好,后期可供参考用于新冠病毒刺突蛋白S1亚基的定量。
Abstract：Aims: This paper aims to establish a quantitative method for the spike glycoprotein S1 subunit of SARS-CoV-2 based on specific peptides produced by trypsin hydrolysis. Methods: Specific peptides were first selected based on the amino acid sequence of spike glycoprotein and its glycosylation modification sites. Reaction parameters for trypsin hydrolysis were optimized to hydrolyze the protein into peptides fully. Finally, a specific peptide-based isotope dilution mass spectrometry was recruited for quantification. Results: We quantified the S1 subunit Mass fraction in a reference sample to be 1.007×10-3; and the expanded uncertainty was 0.070×10-3. It agreed with the result of the amino acid analysis. This method showed good linearity in the range of 0.47×10-9～1×10-3. The method had good reproducibility and correctness with the regression correlation coefficient R2>0.999 9. The intra-day and inter-day deviations were less than 5%; and the accuracy was between 95% and 105%. This method has a low matrix effect and is traceable to the base units of the International System of Units, which is helpful for the quantification of the S1 subunit and the development of reference materials. Conclusions: This method is feasible and performs well as a reference method for quantifying the spike glycoprotein S1 subunit.
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基本信息:
DOI：
中图分类号:R373
引用信息:
[1]马玉清,夏文强,张雅芬等.基于特征肽段定量新冠病毒刺突蛋白S1亚基的方法建立[J].中国计量大学学报,2021,32(04):439-445+459.
基金信息:
国家市场监督管理总局技术保障项目(No.2020YJ058); 浙江省自然科学基金项目(No.LGC21C010001)