Development of Advanced Mass Spectrometric Approaches for the Analysis of the Proteome of Serum from Women with Pre-Eclampsia

蛋白质是细胞的生物机械。当这种机制以某种方式改变时，例如通过修改一个成分，添加或移除形成这个分子工厂的一个单元，这就改变了细胞的运行方式。这些变化是许多疾病的根源。蛋白质的数量和/或性质的变化涉及到的一种疾病状态是妊娠先兆子痫。在这种疾病中，胎盘的血液供应不足会导致一些因子释放到母亲的血液中，其中至少有一些是蛋白质。这些蛋白质破坏母体血管壁的衬里，导致大小血管都变得漏水。血管渗漏增加会导致液体从血管中逸出，从而导致先兆子痫的症状，如高血压、尿蛋白含量增加以及手部和面部肿胀。这些因子在任何可观察到的临床症状之前就会从胎盘中释放出来，目前还没有筛查方法。如果我们能从血浆中识别出这些蛋白质，我们就可以在标准的产前保健制度中对这些蛋白质进行筛查测试。生物液体，如血浆，含有大量不同的蛋白质，所有这些蛋白质的存在水平因个体而异。更重要的是，不同蛋白质的水平彼此之间差别很大。例如，白蛋白存在于非常高的水平(每毫升血浆(10E-3g/ml)数十毫克)，而参与细胞生长的分子白介素6(IL-6)存在于非常低的水平(每毫升(10E-12g/ml)皮克)。使用分析化学方法鉴定此类样品中蛋白质的性质的方法称为蛋白质组学。蛋白质组学工作流程的第一步是使用特定的酶，如从奶牛胰腺提取物中提取的胰酶，将蛋白质消化成小片段，称为多肽。这一步骤将数千种蛋白质的混合物转化为数十万种多肽的混合物。然后，在带电的溶剂流中将多肽喷洒到质谱仪中进行分析。多肽的特性通常是通过将带电的多肽(离子)与惰性气体碰撞来建立的，这会导致它们以可预测的方式解体，从而使我们能够‘读出’多肽序列。这些方法最适合于小肽，因为较大的多肽更灵活，不能有效地碎片。较新的多肽离子测序方法使用电子来诱导碎裂。这些方法对较长的多肽效果最好。本研究将在不同条件下进行蛋白质降解，如不同的酶，以及使用化学物质来阻断酶切位点。将在计算机上模拟酶切割，并选择最佳切割条件进行实验验证。一旦建立了一种方法，这种方法将用于从先兆子痫妇女身上采集的血浆样本，以确定其丰度在“正常”血浆(怀孕的、非先兆子痫的)和疾病发展前后的样本之间发生变化的蛋白质。这将有助于加深对先兆子痫病因的了解，并建立一组标记物，可用于建立妊娠期间血浆样本的预后检测。最终，这将允许对否则会患有先兆子痫的妇女进行识别、流传输和适当的管理。最终的结果将是确定这种危及生命的疾病的药物干预的可能目标。

Proteins are the biological machinery of the cell. When this machinery is altered in some way, for instance via modification of a component, addition or removal of one of the units which form this molecular factory, this changes the way in which the cell performs. Such changes are at the root of many diseases. One disease state where changes in amounts and/ or nature of proteins have been implicated is the pregnancy condition pre-eclampsia. In this disease, a poor blood supply to the placenta induces release of factors, at least some of which are proteins, into the mothers' bloodstream. These proteins damage the lining of walls of maternal blood vessels, causing both small and large blood vessels to become leaky. Increased vessel leakiness leads to fluid escaping from vessels, and thus the symptoms of pre-eclampsia, such as high blood pressure, increased protein content in urine, and swelling of the hands and face. Release of these factors from the placenta occurs before any observable clinical symptoms, and currently no screening methods exist. If we can identify these proteins from blood plasma, we can implement screening tests for these within standard prenatal care regimes. Biological fluids such as plasma contain a huge number of different proteins, all of which are present at levels which vary between individuals. More significantly, the levels of different proteins are very different from one another. For instance, albumin is present at very high levels (tens of milligrams per millilitre of plasma (10E-3g/ml) ), whilst interleukin 6, a molecule involved in cell growth, is present at very low levels (just picograms per millilitre (10E-12g/ml)). Methods to identify the nature of proteins within such samples using analytical chemical methods are termed proteomics. The first step in a proteomic workflow is to digest proteins into small pieces, called peptides, using specific enzymes such as trypsin from cow pancreatic extracts. This step converts a mixture of many thousands of proteins into a mixture of many hundreds of thousands of peptides. Peptides are then analysed by spraying them in a stream of solvent under electrical charge into a mass spectrometer. Identity of the peptides is typically established by colliding the charged peptides (ions) with inert gas, which causes them to fall apart in a predictable manner, allowing us to 'read off' peptide sequence. These methods are best-suited to small peptides, as larger peptides are more flexible and do not fragment efficiently. Newer methods of sequencing peptide ions use electrons to induce fragmentation. These methods work best with longer polypeptides. This study will use proteolysis under different conditions, such as different enzymes and using chemicals to block enzyme cutting sites. Enzymatic cleavage will be simulated on a computer and optimal cleavage conditions selected for experimental substantiation. Once a method has been established, this will be used on plasma samples taken from women with pre-eclampsia to identify proteins whose abundance is altered between 'normal' plasma (pregnant, non-pre-eclamptic) and samples taken prior to and after development of disease. This will enable both increased understanding of the causes of pre-eclampsia and establishment of a panel of markers which can be carried forward for establishment of prognostic testing of plasma samples during pregnancy. Eventually, this will allow identification, streaming and appropriate management of women who would otherwise suffer pre-eclampsia. An eventual outcome will be identification of possible targets for drug intervention in this life-threatening illness.