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 ∼200 attomoles and a limit of quantitation of ∼ 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 × 10⁵ 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.

中文翻译：

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开发平行反应监测 (PRM) 质谱分析法，用于检测 SARS-CoV-2 刺突糖蛋白和核蛋白。

迫切需要在疑似患者样本中检测 SARS-CoV-2 的稳健且高通量的方法，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，但在当前疫情期间，RT-PCR检测的部署极其缓慢，PCR引物、RNA提取试剂盒等关键试剂正处于严重短缺状态。快速护理点病毒抗原检测方法先前已用于诊断呼吸道病毒，例如流感和呼吸道合胞病毒。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，并应探索特异性和灵敏的病毒蛋白检测的替代方法。靶向质谱技术能够以单氨基酸分辨率对特定的蛋白质/肽子集进行鉴定和定量，具有阿托摩尔水平的灵敏度和高重现性。在此，我们报告了一种用于检测相关生物基质中 SARS-CoV-2 刺突蛋白和核蛋白的靶向质谱分析。消化重组全长刺突蛋白和核蛋白，并选择蛋白型肽，使用高分辨率 Orbitrap 仪器进行平行反应监测 (PRM) 定量。生成并评估了一个光谱库，其中包含七个蛋白型肽（四个来自刺突蛋白，三个来自核蛋白）以及前三到四个转换。从原始谱库中，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 病毒粒子的模拟测试样本对测定进行评估，并将其添加到体外衍生的粘液中。PRM 测定的检测限为 ∼200 attomoles，定量限为∼390 attomoles。从测试样本推断，检测 SARS-CoV-2 刺突和核蛋白检测所需的预计病毒颗粒滴度约为 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案。基于质谱的病毒抗原检测方法可能会提供更高的通量，并可作为 RT-PCR 的补充诊断工具。此外，该测定可用于评估存档或最近收集的生物液体、体外研究材料和废水样本中 SARS-CoV-2 的存在。