The detection of SARS-CoV-2 biomarkers by real time PCR (rRT-PCR) has shown that the sensitivity of the test is negatively affected by low viral loads and the severity of the disease. This limitation can be overcome by the use of more sensitive approaches such as mass spectrometry (MS), which has not been explored for the detection of SARS-CoV-2 proteins in saliva. Thus, this study aimed at assessing the translational applicability of mass spectrometry-based proteomics approaches to identify viral proteins in saliva from people diagnosed with COVID-19 within fourteen days after the initial diagnosis, and to compare its performance with rRT-PCR. After ethics approval, saliva samples were self-collected by 42 COVID-19 positive and 16 healthy individuals. Samples from people positive for COVID-19 were collected on average on the sixth day (± 4 days) after initial diagnosis. Viable viral particles in saliva were heat-inactivated followed by the extraction of total proteins and viral RNA. Proteins were digested and then subjected to tandem MS analysis (LC-QTOF-MS/MS) using a data-dependent MS/MS acquisition qualitative shotgun proteomics approach. The acquired spectra were queried against a combined SARS-CoV-2 and human database. The qualitative detection of SARS-CoV-2 specific RNA was done by rRT-PCR. SARS-CoV-2 proteins were identified in all COVID-19 samples (100%), while viral RNA was detected in only 24 out of 42 COVID-19 samples (57.1%). Seven out of 18 SARS-CoV-2 proteins were identified in saliva from COVID-19 positive individuals, from which the most frequent were replicase polyproteins 1ab (100%) and 1a (91.3%), and nucleocapsid (45.2%). Neither viral proteins nor RNA were detected in healthy individuals. Our mass spectrometry approach appears to be more sensitive than rRT-PCR for the detection of SARS-CoV-2 biomarkers in saliva collected from COVID-19 positive individuals up to 14 days after the initial diagnostic test. Based on the novel data presented here, our MS technology can be used as an effective diagnostic test of COVID-19 for initial diagnosis or follow-up of symptomatic cases, especially in patients with reduced viral load.

中文翻译：

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通过转录组学和蛋白质组学分析鉴定唾液中的 SARS-CoV-2 生物标志物

通过实时 PCR (rRT-PCR) 检测 SARS-CoV-2 生物标志物表明，检测的灵敏度受到低病毒载量和疾病严重程度的负面影响。这一限制可以通过使用更灵敏的方法来克服，例如质谱 (MS)，但尚未探索用于检测唾液中 SARS-CoV-2 蛋白的方法。因此，本研究旨在评估基于质谱的蛋白质组学方法的转化适用性，以在初次诊断后 14 天内识别确诊为 COVID-19 的人唾液中的病毒蛋白，并将其性能与 rRT-PCR 进行比较。经伦理批准后，由 42 名 COVID-19 阳性者和 16 名健康者自行采集唾液样本。平均在初次诊断后第六天（± 4 天）收集 COVID-19 阳性人群的样本。将唾液中的活病毒颗粒热灭活，然后提取总蛋白和病毒 RNA。消化蛋白质，然后使用数据依赖型 MS/MS 采集定性鸟枪蛋白质组学方法进行串联 MS 分析 (LC-QTOF-MS/MS)。根据 SARS-CoV-2 和人类数据库对获得的光谱进行查询。通过rRT-PCR对SARS-CoV-2特异性RNA进行定性检测。在所有 COVID-19 样本中均发现了 SARS-CoV-2 蛋白（100%），而在 42 个 COVID-19 样本中仅在 24 个样本中检测到了病毒 RNA（57.1%）。在 COVID-19 阳性个体的唾液中鉴定出 18 种 SARS-CoV-2 蛋白中的 7 种，其中最常见的是复制酶多蛋白 1ab (100%) 和 1a (91.3%) 以及核衣壳 (45.2%)。在健康个体中未检测到病毒蛋白和 RNA。我们的质谱方法似乎比 rRT-PCR 更敏感，可以检测初始诊断测试后 14 天内从 COVID-19 阳性个体收集的唾液中的 SARS-CoV-2 生物标志物。基于此处提供的新数据，我们的 MS 技术可用作 COVID-19 的有效诊断测试，用于对有症状病例的初步诊断或随访，特别是对于病毒载量减少的患者。