Absolute quantification of SARS-CoV-2 proteins and their human targets for informing drug strategies and accelerating vaccine development

SARS-CoV-2 蛋白及其人类靶标的绝对定量，为药物策略提供信息并加速疫苗开发

• 批准号: BB/V013866/1
• 负责人: Martin Buck
• 金额: $ 40.82万
• 依托单位: Imperial College London
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2020
• 资助国家: 英国
• 起止时间: 2020 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FV013866%2F1
• 关键词: Absolute; quantification; SARS; CoV; proteins

Understanding how the SARS-CoV-2 virus is infectious and causes disease requires a deep understanding of how the infectious particle-the virus- assembles from its component parts, and can make copies of itself and then invade cells and humans. We plan to work out how its building blocks-its proteins in particular-are produced, in what form and how much of each is made. To do so, we will use a method that weighs a protein, and counts how many proteins are in a sample from cells infected with the virus. These numbers will inform strategies for vaccine development as well as offer a deeper understanding of how the virus forms.We will use a method called Multiple-Reaction-Monitoring Mass Spectrometry (MRM-MS) with protein standards which we will manufacture in our laboratory. This methodology allows to directly measure absolute protein concentrations in complex biological samples. Our approach has great scope for upscaling, because any drug and vaccine development could adopt and benefit from our methodology.We propose to produce bespoke labelled synthetic protein standards and measure the absolute SARS-CoV-2 proteome, including some post-translational modifications, of (i) the virion (likely 9 proteins); (ii) within infected human cell lines (about viral 29 proteins), (iii) including yet undetected proteins; (iv) alongside key human proteins (19 proteins) the virus interacts with and or are considered drug targets; (v) determine the antigen-antibody ratios following vaccination trials in mice and humans and (vi) provide said standards to other laboratories to enable them to conduct similar analytics.

要了解SARS-CoV-2病毒如何具有传染性并导致疾病，需要深入了解传染性颗粒-病毒-如何从其组成部分组装，并可以复制自己，然后入侵细胞和人类。我们计划弄清楚它的组成部分，特别是蛋白质是如何产生的，以什么形式产生，以及每种蛋白质产生的量。为了做到这一点，我们将使用一种方法来衡量蛋白质，并计算感染病毒的细胞样本中有多少蛋白质。这些数字将为疫苗开发策略提供信息，并提供对病毒如何形成的更深入了解。我们将使用一种称为多反应监测质谱法（MRM-MS）的方法，使用我们实验室制造的蛋白质标准品。该方法允许直接测量复杂生物样品中的绝对蛋白质浓度。我们的方法有很大的升级空间，因为任何药物和疫苗的开发都可以采用我们的方法并从中受益。我们建议生产定制的标记合成蛋白标准品，并测量SARS-CoV-2的绝对蛋白质组，包括一些翻译后修饰，（i）病毒粒子（可能9种蛋白质）;（ii）在感染的人细胞系内（关于病毒29蛋白），（iii）包括尚未检测到的蛋白;（iv）与关键的人类蛋白质一起（19种蛋白质）病毒与药物相互作用或被认为是药物靶点;（V）在小鼠和人中进行疫苗接种试验后确定抗原-抗体比率，以及将所述标准提供给其他实验室，使他们能够进行类似的分析。