Mass Spectrometric Analysis of Urine from COVID-19 Patients for Detection of SARS-CoV‑2 Viral Antigen and to Study Host Response

SARS-CoV-2 infection has become a major public health burden and affects many organs including lungs, kidneys, the liver, and the brain. Although the virus is readily detected and diagnosed using nasopharyngeal swabs by reverse transcriptase polymerase chain reaction (RT-PCR), detection of its presence in body fluids is fraught with difficulties. A number of published studies have failed to detect viral RNA by RT-PCR methods in urine. Although microbial identification in clinical microbiology using mass spectrometry is undertaken after culture, here we undertook a mass spectrometry-based approach that employed an enrichment step to capture and detect SARS-CoV-2 nucleocapsid protein directly from urine of COVID-19 patients without any culture. We detected SARS-CoV-2 nucleocapsid protein-derived peptides from 13 out of 39 urine samples. Further, a subset of COVID-19 positive and COVID-19 negative urine samples validated by mass spectrometry were used for the quantitative proteomics analysis. Proteins with increased abundance in urine of SARS-CoV-2 positive individuals were enriched in the acute phase response, regulation of complement system, and immune response. Notably, a number of renal proteins such as podocin (NPHS2), an amino acid transporter (SLC36A2), and sodium/glucose cotransporter 5 (SLC5A10), which are intimately involved in normal kidney function, were decreased in the urine of COVID-19 patients. Overall, the detection of viral antigens in urine using mass spectrometry and alterations of the urinary proteome could provide insights into understanding the pathogenesis of COVID-19.严重急性呼吸综合征冠状病毒2（SARS-CoV-2）感染已成为重大公共卫生负担，可累及肺、肾、肝、脑等多个器官。尽管可通过鼻咽拭子采样结合逆转录聚合酶链反应（reverse transcriptase polymerase chain reaction, RT-PCR）快速检测并诊断该病毒，但在体液中检测其存在却困难重重。多项已发表的研究均未能通过RT-PCR法在尿液中检测到病毒RNA。尽管临床微生物学领域中基于质谱法（mass spectrometry）的微生物鉴定需在培养后进行，但本研究采用了一种基于质谱法的方法，该方法无需培养步骤，仅通过富集步骤即可直接从新型冠状病毒肺炎（COVID-19）患者的尿液中捕获并检测SARS-CoV-2核衣壳蛋白。我们在39份尿液样本中的13份里检测到了SARS-CoV-2核衣壳蛋白衍生肽段。此外，我们选取经质谱法验证的COVID-19阳性及阴性尿液样本亚组，进行定量蛋白质组学分析。SARS-CoV-2感染者尿液中丰度上调的蛋白质，显著富集于急性期反应、补体系统调控及免疫应答相关通路。值得注意的是，多种与正常肾功能密切相关的肾脏相关蛋白在COVID-19患者尿液中表达下调，例如足孔蛋白（podocin, NPHS2）、氨基酸转运蛋白（SLC36A2）以及钠/葡萄糖协同转运蛋白5（SLC5A10）。总体而言，通过质谱法检测尿液中的病毒抗原，以及尿液蛋白质组的变化，可为阐明COVID-19的发病机制提供新的见解。