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

开发用于分析先兆子痫女性血清蛋白质组的先进质谱方法

• 批准号: EP/E043143/2
• 负责人: Sarah Hart
• 金额: --
• 依托单位: Keele University
• 依托单位国家: 英国
• 项目类别: Fellowship
• 财政年份: 2009
• 资助国家: 英国
• 起止时间: 2009 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=EP%2FE043143%2F2
• 关键词: Development; Advanced; Mass; Spectrometric; Approaches

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.

蛋白质是细胞的生物机器。当这种机制以某种方式改变时，例如通过修改一种成分，添加或删除形成这种分子工厂的一个单元，这会改变细胞的表现方式。这种变化是许多疾病的根源。其中涉及蛋白质的量和/或性质的变化的一种疾病状态是妊娠状况先兆子痫。在这种疾病中，胎盘的血液供应不足导致因子释放，其中至少有一些是蛋白质，进入母亲的血液。这些蛋白质破坏母体血管壁的内层，导致大小血管都变得渗漏。血管渗漏增加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、尿中蛋白质含量增加以及手和脸肿胀。这些因子从胎盘中的释放发生在任何可观察到的临床症状之前，并且目前不存在筛查方法。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护理制度中对这些蛋白质进行筛查。生物液体，如血浆，含有大量不同的蛋白质，所有这些蛋白质都以个体之间不同的水平存在。更重要的是，不同蛋白质的水平彼此非常不同。例如，白蛋白以非常高的水平存在（每毫升血浆数十毫克（10 E-3g/ml）），而白细胞介素6，一种参与细胞生长的分子，以非常低的水平存在（每毫升仅皮克（10 E-12 g/ml））。使用分析化学方法鉴定此类样本中蛋白质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（如来自牛胰腺提取物的胰蛋白酶）将蛋白质消化成小片段（称为肽）。这一步骤将成千上万种蛋白质的混合物转化为数十万种肽的混合物。然后通过将它们在带电的溶剂流中喷射到质谱仪中来分析肽。肽的身份通常是通过将带电的肽（离子）与惰性气体碰撞来确定的，这会导致它们以可预测的方式分解，从而使我们能够“读取”肽序列。这些方法最适合小肽，因为较大的肽更灵活，不能有效地片段化。较新的肽离子测序方法使用电子诱导片段化。这些方法对较长的多肽效果最好。本研究将采用不同条件下的蛋白质水解，如不同的酶和使用化学物质阻断酶切位点。将在计算机上模拟酶促裂解，并选择最佳裂解条件用于实验证实。一旦建立了一种方法，这将用于从患有先兆子痫的女性中采集的血浆样本，以鉴定其丰度在“正常”血浆（妊娠，非先兆子痫）和疾病发展前后采集的样本之间发生变化的蛋白质。这将使得能够增加对先兆子痫的原因的理解，并建立一组标记物，其可以用于建立妊娠期间血浆样品的预后测试。最终，这将允许识别，分流和适当管理否则将遭受先兆子痫的妇女。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。

项目成果

Electron transfer dissociation of native peptides facilitates enhanced identification of urinary peptides

• DOI: 10.1016/j.ijms.2015.08.025
• 发表时间: 2015-11
• 期刊: International Journal of Mass Spectrometry
• 影响因子: 1.8
• 作者: S. Hart;L. Kenny;J. Myers;P. Baker

[203-POS]

[203-POS]

• DOI: 10.1016/j.preghy.2014.10.209
• 发表时间: 2015
• 期刊: An International Journal of Women's Cardiovascular Health
• 作者: Hart S

[82-OR]

[82-或]

• DOI: 10.1016/j.preghy.2014.10.086
• 发表时间: 2015
• 期刊: An International Journal of Women's Cardiovascular Health
• 作者: Hart S

Electron Transfer Dissociation Analysis of the Urinary Peptidome in Pregnancy

妊娠期尿肽组的电子转移解离分析

• 发表时间: 2009
• 作者: Richard Blankley