ProGenTomics, Laboratory of Pharmaceutical Biotechnology, Ghent University, 9000 Ghent, Belgium.
Laboratory of Toxicology, Department of Bioanalysis, Faculty of Pharmaceutical Sciences, Ghent University, 9000 Ghent, Belgium.
Anal Chem. 2022 Dec 20;94(50):17379-17387. doi: 10.1021/acs.analchem.2c01610. Epub 2022 Dec 9.
The pandemic readiness toolbox needs to be extended, targeting different biomolecules, using orthogonal experimental set-ups. Here, we build on our Cov-MS effort using LC-MS, adding SISCAPA technology to enrich proteotypic peptides of the SARS-CoV-2 nucleocapsid (N) protein from trypsin-digested patient samples. The CovMS assay is compatible with most matrices including nasopharyngeal swabs, saliva, and plasma and has increased sensitivity into the attomole range, a 1000-fold improvement compared to direct detection in a matrix. A strong positive correlation was observed with qPCR detection beyond a quantification cycle of 30-31, the level where no live virus can be cultured. The automatable sample preparation and reduced LC dependency allow analysis of up to 500 samples per day per instrument. Importantly, peptide enrichment allows detection of the N protein in pooled samples without sensitivity loss. Easily multiplexed, we detect variants and propose targets for Influenza A and B detection. Thus, the CovMS assay can be adapted to test for many different pathogens in pooled samples, providing longitudinal epidemiological monitoring of large numbers of pathogens within a population as an early warning system.
疫情准备工具包需要扩展，针对不同的生物分子，使用正交实验设置。在这里，我们在使用 LC-MS 的 Cov-MS 工作基础上，添加 SISCAPA 技术，从胰蛋白酶消化的患者样本中富集 SARS-CoV-2 核衣壳 (N) 蛋白的表位肽。CovMS 检测法与大多数基质兼容，包括鼻咽拭子、唾液和血浆，其灵敏度提高到飞摩尔范围，与直接在基质中检测相比提高了 1000 倍。在 qPCR 检测超过 30-31 个定量循环时，观察到与 qPCR 检测有很强的正相关性，此时无法培养活病毒。自动化的样品制备和减少对 LC 的依赖允许每个仪器每天分析多达 500 个样本。重要的是，肽富集允许在没有灵敏度损失的情况下对混合样本中的 N 蛋白进行检测。我们很容易实现多重检测，并提出了用于检测甲型和乙型流感的目标。因此，CovMS 检测法可以适应混合样本中多种不同病原体的检测，为人群中大量病原体提供纵向流行病学监测，作为早期预警系统。
