National Center for Environmental Health, Centers for Disease Control and Prevention, Atlanta, GA 30341, USA.
Oak Ridge Institute for Science and Education, Centers for Disease Control and Prevention, Atlanta, GA 30341, USA.
Vaccine. 2021 Aug 23;39(36):5106-5115. doi: 10.1016/j.vaccine.2021.07.066. Epub 2021 Jul 24.
The emergence and subsequent global outbreak of the novel coronavirus SARS-CoV-2 prompted our laboratory to launch efforts to develop methods for SARS-CoV-2 antigen detection and quantification. We present an isotope dilution mass spectrometry method (IDMS) for rapid and accurate quantification of the primary antigens, spike and nucleocapsid proteins. This IDMS method utilizes liquid chromatography-tandem mass spectrometry (LC-MS/MS) to analyze sample tryptic digests for detection and quantification of selected conserved peptides of SARS-CoV-2 spike and nucleocapsid proteins. The IDMS method has the necessary attributes to be successfully utilized for accurate quantification in SARS-CoV-2 protein-based vaccines and as targets of rapid diagnostic tests. Absolute quantification was achieved by quantifying and averaging 5 peptides for spike protein (3 peptides in the S1 subunit and 2 peptides in the S2 subunit) and 4 peptides for nucleocapsid protein. The overall relative standard deviation of the method was 3.67% for spike protein and 5.11% for nucleocapsid protein. IDMS offers speed (5 h total analysis time), sensitivity (LOQ; 10 fmol/µL) and precision for quantification of SARS-CoV-2 spike and nucleocapsid proteins.
新型冠状病毒 SARS-CoV-2 的出现及其随后在全球的爆发促使我们实验室着手开发用于 SARS-CoV-2 抗原检测和定量的方法。我们提出了一种用于快速准确定量主要抗原（刺突和核衣壳蛋白）的同位素稀释质谱法（IDMS）。该 IDMS 方法利用液相色谱-串联质谱（LC-MS/MS）分析样品胰蛋白酶消化物，以检测和定量 SARS-CoV-2 刺突和核衣壳蛋白的选定保守肽段。该 IDMS 方法具有成功用于 SARS-CoV-2 基于蛋白的疫苗的准确定量以及快速诊断测试的目标的必要属性。通过对 5 个刺突蛋白（S1 亚基中的 3 个肽和 S2 亚基中的 2 个肽）和 4 个核衣壳蛋白的肽进行定量和平均，实现了绝对定量。该方法的总体相对标准偏差为刺突蛋白的 3.67%，核衣壳蛋白的 5.11%。IDMS 为 SARS-CoV-2 刺突和核衣壳蛋白的定量提供了速度（总分析时间 5 小时）、灵敏度（LOQ；10 fmol/μL）和精密度。
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