Cov²MS: an automated matrix-independent assay for mass spectrometric detection and measurement of SARS-CoV-2 nucleocapsid protein

Introduction. The pandemic readiness toolbox needs to be extended, providing diagnostic tools that target different biomolecules, using orthogonal experimental setups and fit-for-purpose specification of detection. Here, we build on a previous Cov-MS effort to use liquid chromatography coupled to mass spectrometry (LC-MS) and describe a method developed to allow accurate, high throughput measurement of SARS-CoV-2 nucleocapsid (NCAP) protein.
Methods. We use Stable Isotope Standards and Capture by Anti-Peptide Antibodies (SISCAPA) technology to enrich and quantify proteotypic peptides of the NCAP protein from trypsin-digested samples from COVID-19 patients.
Results. This makes the Cov²MS assay compatible with most matrices including nasopharyngeal swabs, saliva and blood plasma, while increasing the sensitivity into the attomole range, up to a 1000-fold increase compared to direct detection in matrix. In addition, there is a strong positive correlation between the SISCAPA antigen assay and qPCR detection up to a quantification cycle (Cq) of 30. The automatable “addition only” sample preparation and digestion protocol, the peptide enrichment and the reduced dependency upon LC together allow analysis of up to 500 samples per day per MS instrument. Importantly, peptide enrichment allowed detection of NCAP protein in a pooled sample containing a single PCR positive patient mixed with 31 PCR negative samples, without loss in sensitivity. We also propose novel target peptides for Influenza A and B.
Conclusion. Since the Cov²MS assay is insensitive to matrix or pooling and easily multiplexed, it can be used to test for many different pathogens and could provide longitudinal epidemiological monitoring of large numbers of pathogens within a population, applied as an early warning system.

引言。当前的大流行应对工具箱亟待拓展，亟需开发针对不同生物分子、采用正交实验设计且符合适配用途检测规范的诊断工具。本研究基于此前的Cov-MS工作，采用液相色谱-质谱联用（liquid chromatography coupled to mass spectrometry, LC-MS）技术，开发了一种可精准、高通量检测严重急性呼吸综合征冠状病毒2型（SARS-CoV-2）核衣壳（nucleocapsid, NCAP）蛋白的方法。

方法。本研究采用抗肽抗体捕获稳定同位素标准（Stable Isotope Standards and Capture by Anti-Peptide Antibodies, SISCAPA）技术，对新冠患者胰蛋白酶消化样本中的核衣壳蛋白蛋白组特异性肽段进行富集与定量分析。

结果。本优化后的Cov²MS检测方法可兼容绝大多数样本基质，包括鼻咽拭子、唾液与血浆，且将检测灵敏度提升至阿托摩尔级，较基质中直接检测的灵敏度提升最高可达1000倍。此外，在定量循环数（quantification cycle, Cq）不超过30的范围内，SISCAPA抗原检测与qPCR检测结果呈显著正相关。该流程具备自动化适配性，采用仅需添加试剂的样本前处理与酶解方案、肽段富集步骤，且对液相色谱的依赖度降低，可实现单台质谱仪器每日最多处理500份样本。值得注意的是，肽段富集技术可在混合样本中检出核衣壳蛋白：该混合样本由1份PCR阳性样本与31份PCR阴性样本混合而成，且未出现灵敏度损失。本研究同时提出了针对甲型流感病毒与乙型流感病毒的新型靶向肽段。

结论。由于Cov²MS检测方法不受样本基质与混合操作的影响，且易于实现多重检测，因此可用于检测多种不同病原体，能够对人群中大量病原体开展纵向流行病学监测，作为早期预警系统加以应用。