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

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，并复制自身，然后侵入细胞和人体。我们计划弄清楚它的组成部分——尤其是蛋白质——是如何产生的，以什么形式产生，每种成分有多少。为此，我们将使用一种方法，对蛋白质进行称重，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战略提供信息，并使人们更深入地了解病毒是如何形成的。我们将使用一种称为多重反应监测质谱法（MRM-MS）的方法，我们将在我们的实验室制造蛋白质标准。这种方法可以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并受益于我们的方法。我们建议生产定制的标记合成蛋白质标准，并测量绝对的SARS-CoV-2蛋白质组，包括一些翻译后修饰，(i)病毒粒子（可能是9种蛋白质）；（二）在受感染的人类细胞系内（约29种病毒蛋白），（三）包括尚未检测到的蛋白；（四）病毒与关键人类蛋白（19种蛋白）相互作用或被认为是药物靶点；确定在小鼠和人类接种疫苗试验后的抗原-抗体比率，并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.