Bate Neil, Lane Dan, Evans Sian E, Salim Farah, Allcock Natalie S, Haigh Richard, Sale Julian E, Jones Donald J L, Brindle Nicholas P J
Department of Cardiovascular Sciences, University of Leicester, University Road, Leicester, Leicester LE1 7RH, U.K.
van Geest MS-OMICS Facility, University of Leicester, University Road, Leicester, Leicester LE1 7RH, U.K.
JACS Au. 2025 Feb 4;5(2):747-755. doi: 10.1021/jacsau.4c00980. eCollection 2025 Feb 24.
Mass spectrometry (MS) is a potentially powerful approach for the diagnostic detection of SARS-CoV-2 and other viruses. However, MS detection is compromised when viral antigens are present at low concentrations, especially in complex biological media. We hypothesized that viral receptors could be used for viral target capture to enable detection by MS under such conditions. This was tested using the extracellular domain of the SARS-CoV-2 receptor ACE2. To maximize recovery of the target protein, directed protein evolution was first used to increase the affinity of ACE2 for spike protein. This generated an evolved ACE2 with increased binding affinity for the spike protein receptor-binding domain (RBD). However, as with other affinity-enhanced evolved forms of ACE2, binding was sensitive to mutations in variant RBDs. As an alternative strategy to maximize capture, the native ACE2 extracellular domain was engineered for increased binding by the addition of an oligomerization scaffold to create pentameric ACE2. This bound extremely tightly to SARS-CoV-2 RBD, with an increase in apparent affinity of several thousand-fold over monomeric ACE2, and RBD retention of more than 8 h. Immobilization of multimeric ACE2 enabled quantitative enrichment of viral spike protein from saliva and increased the sensitivity of detection by MS. These data show that capture by engineered receptors combined with MS can be an effective, rapid method for detection and quantitation of target protein. A similar approach could be used for attachment proteins of other viruses or any target protein for which there are suitable receptors.
质谱分析（MS）是一种用于诊断检测严重急性呼吸综合征冠状病毒2（SARS-CoV-2）及其他病毒的潜在强大方法。然而，当病毒抗原以低浓度存在时，尤其是在复杂生物介质中，质谱检测会受到影响。我们推测病毒受体可用于病毒靶点捕获，以便在这种情况下通过质谱进行检测。我们使用SARS-CoV-2受体血管紧张素转换酶2（ACE2）的胞外域对此进行了测试。为了最大限度地回收目标蛋白，首先使用定向蛋白质进化来提高ACE2对刺突蛋白的亲和力。这产生了一种对刺突蛋白受体结合域（RBD）具有更高结合亲和力的进化型ACE2。然而，与其他亲和力增强的ACE2进化形式一样，其结合对变异RBD中的突变敏感。作为最大化捕获的替代策略，通过添加寡聚支架对天然ACE2胞外域进行工程改造，以增加其结合能力，从而产生五聚体ACE2。这种五聚体ACE2与SARS-CoV-2 RBD紧密结合，表观亲和力比单体ACE2增加了数千倍，并且RBD保留时间超过8小时。固定化的多聚体ACE2能够从唾液中定量富集病毒刺突蛋白，并提高了质谱检测的灵敏度。这些数据表明，工程化受体捕获与质谱联用可以成为一种有效、快速的目标蛋白检测和定量方法。类似方法可用于其他病毒的附着蛋白或任何存在合适受体的目标蛋白。
