Double standards in quantitative proteomics: Development of calibrators for multiplexed quantitative western blotting or mass spectrometry

生物学研究在很大程度上是一门量化科学，在生物分子科学中，始终需要能够以高度敏感的方式测量特定分子的数量及其丰度的变化。此外，生物分子必须在成千上万个相似的生物分子的背景下进行测量，因此测量必须具有选择性和敏感性。其中之一依赖于一种非常昂贵和极其复杂的仪器--质谱仪，并需要高度熟练的专业人员来提供为该平台开发的非常复杂的分析。第二种方法使用非常便宜的实验室设备(可能在几乎每个实验室都能找到)，依靠抗体“找到”并测量感兴趣的生物分子--这是一颗测量的神奇子弹。然而，许多实验室采用的抗体方法并不是真正的定量方法。他们依赖于将生物分子在一种松散的凝胶中分离出来，然后在使用抗体找到感兴趣的生物分子之前，将生物分子‘印迹’到特氟龙膜上--也就是所谓的‘蛋白质印迹’。西方片杂乱无章，变化无常，容易出错--正如有人评论的那样，西方片可以是好的，也可以是坏的，也可以是丑的！为了绕过手动Western印迹的问题，一种负担得起的商业仪器已经上市，在我们手中，它的性能至少能够与质谱仪一样好。我们相信，这样的仪器具有巨大的潜力，可以通过西风将免疫学定量带入与质谱学相同的标准。根据我们的经验，这两种方法(自动抗体检测或质谱学)可以同样好地执行，但在某些情况下，结果并不一致。其中一个问题是，我们对这两种方法都缺乏合适的标准，这阻碍了直接比较。为了解决这个问题，我们发明了在地球上从未见过的人造蛋白质(DOSCATs)的概念，它是通过基因设计创造出来的，以执行特定的功能-提供标准蛋白质，这些蛋白质可以同样好地用于免疫学或质谱学定量。这一创新的概念将在两条量化途径之间建立无缝连接，同时将加强量化，以便准确计算生物样本中特定分子的数量。由于这是一项技术开发应用，我们将使用的测试系统之所以被选择，是因为我们非常了解它们，并了解它们在自然系统中的表现。这减少了我们用质谱仪或Western blotting进行量化时的不确定性。我们将开发针对不同领域的DOSCATs，如奶牛乳房炎(具有福利和商业后果)、在一个旨在了解和制定加强啮齿动物控制策略的项目中对非常相似的生物分子(称为异构体)进行单独量化，以及一个项目的目标是向试图建立生命过程模型的计算生物学家提供准确的定量数据。

Biological research is a largely quantitative science, and in biomolecular sciences, there is an ever-present need to be able to measure the amounts of specific molecules, and changes in their abundance, in a highly sensitive fashion. Moreover, the biomolecules have to be measured in a background of many tens of thousands of similar biomolecules, and thus, the measurements must be selective as well as sensitive.There are two analytical approaches that are commonly used to count the numbers of biomolecules in a system. One relies on a very expensive and extremely sophisticated instrument, a mass spectrometer, and requires highly skilled specialist staff to deliver the very complex assays that are developed for this platform. The second approach uses very inexpensive laboratory equipment (likely to be found in almost every lab) and relies on antibodies to 'find' and measure the biomolecule of interest - a magic bullet for measurement. However, antibody methods, as practiced in many labs, are not really quantitative. They rely on separation of biomolecules in a sloppy gel, and then 'blotting' the biomolecules onto a teflon membrane before using antibodies to 'find; the biomolecule of interest -so called 'western blots'. Western blots are messy, highly variable and error prone - as has been commented, westerns can be good, bad, or ugly! To circumvent the problems of manual western blots, an affordable commercially available instrument has come to market that, in our hands, is capable of performance at least as good as a mass spectrometer. We believe that instrument such as this has huge potential to bring immunological quantification via westerns to the same standard as mass spectrometry.In our experience, the two methods (automated antibody detection or mass spectrometry) can be made to perform equally well, but in some circumstances, the results do not agree. One of the problems is that we lack suitable standards for both methods, which prevents direct comparison. To solve this problem, we have invented the concept of artificial proteins (DOSCATs) , never previously seen on the planet, that are created through gene design to perform a specific function - providing standard proteins that can be used equally well for immunological or mass spectrometric quantification. This innovative concept will create a seamless connection between the two routes to quantification, and at the same time, will enhance the quantification to permit accurate counting of the numbers of specific molecules in a biological sample.Because this is a technological development application, the test systems that we will use are selected because we know them well, and know how they behave in natural systems. This reduces uncertainty when we come to perform quantification by mass spectrometry or western blotting. We will develop DOSCATs that address diverse areas such as mastitis in dairy cattle (which has both welfare and commercial consequences), separate quantification of very similar biomolecules (called isoforms) in a project that aims to understand and develop strategies for enhanced rodent control and a programme that has the goal of providing precise quantitative data to computational biologists who are trying to build models of living process.