vPro-MS enables identification of human-pathogenic viruses from patient samples by untargeted proteomics

Viral infections are commonly diagnosed by the detection of viral genome fragments or proteins using targeted methods such as PCR and immunoassays. In contrast, metagenomics enables the untargeted identification of viral genomes, expanding its applicability across a broader spectrum. In this study, we introduce proteomics as a complementary approach for the untargeted identification of human-pathogenic viruses from patient samples. The viral proteomics workflow (vPro-MS) is based on an in-silico derived peptide library covering the human virome in UniProtKB (331 viruses, 20,386 genomes, 121,977 peptides), which was especially designed for diagnostic purposes. A scoring algorithm (vProID score) was developed to assess the confidence of virus identification from proteomics data. In combination with high-throughput diaPASEF-based data acquisition, this workflow enables the analysis of up to 60 samples per day. The specificity was determined to be > 99,9 % in an analysis of 221 plasma, swab and cell culture samples covering 18 different viruses (e.g. SARS, MERS, EBOV, MPXV). The sensitivity of this approach for the detection of SARS-CoV-2 in nasopharyngeal swabs corresponds to a PCR cycle threshold of 27 with comparable quantitative accuracy to metagenomics. vPro-MS enables the integration of untargeted virus identification in large-scale proteomic studies of biofluids such as human plasma to detect previously undiscovered virus infections in patient specimens.

病毒感染通常可通过聚合酶链式反应（PCR）、免疫测定等靶向方法检测病毒基因组片段或蛋白质实现诊断。与此形成对比的是，宏基因组学（metagenomics）可实现非靶向的病毒基因组鉴定，其应用场景更为广泛。本研究中，我们提出将蛋白质组学作为一种补充方法，用于从患者样本中非靶向鉴定人类致病病毒。该病毒蛋白质组学工作流程（vPro-MS）基于计算机模拟构建的肽库，该肽库覆盖UniProtKB数据库中的人类病毒组（包含331种病毒、20386个基因组、121977条肽段），专为诊断用途设计。研究开发了一种评分算法（vProID score），用于评估蛋白质组学数据中病毒鉴定结果的置信度。结合基于高通量diaPASEF的数据采集技术，该工作流程每日可分析多达60个样本。在对涵盖18种不同病毒（如严重急性呼吸综合征病毒SARS、中东呼吸综合征病毒MERS、埃博拉病毒EBOV、猴痘病毒MPXV）的221份血浆、拭子及细胞培养样本的分析中，该方法的特异性大于99.9%。该方法在鼻咽拭子样本中检测严重急性呼吸综合征冠状病毒2型（SARS-CoV-2）的灵敏度对应聚合酶链式反应循环阈值为27，且定量准确性与宏基因组学相当。vPro-MS可实现在人类血浆等生物流体的大规模蛋白质组学研究中整合非靶向病毒鉴定，从而在患者样本中检测此前未被发现的病毒感染。