Development of a Parallel Reaction Monitoring Mass Spectrometry Assay for the Detection of SARS-CoV-2 Spike Glycoprotein and Nucleoprotein
Development of a Parallel Reaction Monitoring Mass Spectrometry Assay for the Detection of SARS-CoV-2 Spike Glycoprotein and Nucleoprotein
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DOI：
10.1021/acs.analchem.0c02288
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发表时间：
2020-10-20
影响因子：
7.4
通讯作者：
Hess, Sonja
中科院分区：
文献类型：
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作者：
Cazares, Lisa H.;Chaerkady, Raghothama;Hess, Sonja
目前迫切需要稳健和高通量的方法来检测疑似患者样本中的SARS-CoV-2，以促进疾病管理、监测和控制。尽管逆转录聚合酶链反应（RT-PCR）等核酸检测方法是金标准，但在当前大流行期间，RT-PCR检测的部署极其缓慢，PCR引物和RNA提取试剂盒等关键试剂处于严重短缺状态。快速床旁病毒抗原检测方法先前已用于诊断呼吸道病毒，如流感病毒和呼吸道合胞病毒。因此，直接检测患者样本中的SARS-CoV-2病毒抗原也可用于诊断活动性感染，并应探索特异性和敏感性病毒蛋白检测的替代方法。靶向质谱技术已经能够以阿托摩尔水平的灵敏度和高再现性以单个氨基酸分辨率鉴定和定量蛋白质/肽的限定子集。在此，我们报告了一种靶向质谱检测SARS-CoV-2刺突蛋白和核蛋白在相关的生物基质。消化重组全长刺突蛋白和核蛋白，并选择蛋白型肽用于使用高分辨率Orbitrap仪器进行平行反应监测（PRM）定量。一个光谱库，其中包含7个蛋白质型肽（四个从刺突蛋白和三个从核蛋白）和前三到四个转换，生成和评价。从原始光谱库中，我们选择了两种性能最佳的肽用于最终的PRM测定。使用含有灭活SARS-CoV-2病毒粒子的模拟试验样品（添加到体外来源的粘液中）对该测定进行评价。PRM测定提供了类似于200阿托摩尔的检测限和类似于390阿托摩尔的定量限。从测试样品外推，检测SARS-CoV-2加标和核蛋白检测所需的病毒颗粒的预计滴度约为2 × 10（5）病毒颗粒/mL，使其成为RT-PCR检测的有吸引力的替代方法。潜在地，用于病毒抗原检测的基于质谱的方法可以提供更高的通量，并且可以作为RT-PCR的补充诊断工具。此外，该检测方法可用于评估存档或最近收集的生物液体，体外衍生的研究材料和废水样品中SARS-CoV-2的存在。
There is an urgent need for robust and high-throughput methods for SARS-CoV-2 detection in suspected patient samples to facilitate disease management, surveillance, and control. Although nucleic acid detection methods such as reverse transcription polymerase chain reaction (RT-PCR) are the gold standard, during the current pandemic, the deployment of RT-PCR tests has been extremely slow, and key reagents such as PCR primers and RNA extraction kits are at critical shortages. Rapid point-of-care viral antigen detection methods have been previously employed for the diagnosis of respiratory viruses such as influenza and respiratory syncytial viruses. Therefore, the direct detection of SARS-CoV-2 viral antigens in patient samples could also be used for diagnosis of active infection, and alternative methodologies for specific and sensitive viral protein detection should be explored. Targeted mass spectrometry techniques have enabled the identification and quantitation of a defined subset of proteins/peptides at single amino acid resolution with attomole level sensitivity and high reproducibility. Herein, we report a targeted mass spectrometry assay for the detection of SARS-CoV-2 spike protein and nucleoprotein in a relevant biological matrix. Recombinant full-length spike protein and nucleoprotein were digested and proteotypic peptides were selected for parallel reaction monitoring (PRM) quantitation using a high-resolution Orbitrap instrument. A spectral library, which contained seven proteotypic peptides (four from spike protein and three from nucleoprotein) and the top three to four transitions, was generated and evaluated. From the original spectral library, we selected two best performing peptides for the final PRM assay. The assay was evaluated using mock test samples containing inactivated SARS-CoV-2 virions, added to in vitro derived mucus. The PRM assay provided a limit of detection of similar to 200 attomoles and a limit of quantitation of similar to 390 attomoles. Extrapolating from the test samples, the projected titer of virus particles necessary for the detection of SARS-CoV-2 spike and nucleoprotein detection was approximately 2 x 10(5) viral particles/mL, making it an attractive alternative to RT-PCR assays. Potentially, mass spectrometry-based methods for viral antigen detection may deliver higher throughput and could serve as a complementary diagnostic tool to RT-PCR. Furthermore, this assay could be used to evaluate the presence of SARS-CoV-2 in archived or recently collected biological fluids, in vitro-derived research materials, and wastewater samples.