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

要了解SARS-CoV-2病毒是如何具有传染性并导致疾病的，需要深入了解传染性颗粒--病毒--是如何从其组成部分组装起来的，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成分--特别是它的蛋白质--是如何产生的，是以什么形式产生的，以及每一种成分的量是多少。为此，我们将使用一种称重蛋白质的方法，并计算来自感染病毒的细胞的样本中有多少蛋白质。这些数字将为疫苗开发战略提供参考，并提供对病毒如何形成的更深层次的了解。我们将使用一种名为多反应监测质谱学(MRM-MS)的方法和我们将在实验室制造的蛋白质标准。这种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推广空间，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质标准，并测量SARS-CoV-2蛋白质组的绝对，包括一些翻译后修饰，(I)病毒粒子(可能是9个蛋白质)；(Ii)在受感染的人类细胞系内(约29个蛋白质)，(Iii)包括尚未检测到的蛋白质；(Iv)连同关键的人类蛋白质(19个蛋白质)病毒与病毒相互作用和/或被认为是药物靶标；(V)在小鼠和人类进行疫苗接种试验后确定抗原-抗体比率，并(Vi)向其他实验室提供上述标准，使它们能够进行类似的分析。

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.