Mining Complex Human Fluids for New Potential Protein Biomarkers

 描述(申请人提供)：在这项提案中，我们试图展示一种新的实验方法，以开发一种新的差减方法来从复杂的人体体液样本中去除丰富的蛋白质。最终目标是开发一套方法或工具，以促进从头发现一套完整的低丰度潜在蛋白质生物标志物，从中可以进行更有针对性的研究。人们特别感兴趣的是发现潜在的癌症生物标记物，在过去，我们研究了肺癌样本和来自同一队列的参考文献。在这个第一阶段的提案中，我们试图展示选择性和特异性地从人血浆中去除人血清白蛋白异构体和二聚体。这些血清白蛋白占人血清或血浆中蛋白质的一半，在串联质谱学和差示凝胶电泳法中都会引起非常严重的问题。第二阶段将扩展到去除三个数量级的丰富蛋白质的工作，比现有最先进的方法增加了50倍。一个关键的特点是，选择这些条件是为了最大限度地减少蛋白质之间的相互作用，这会损害现有方法的性能。生产的简化人体体液样本将与2D凝胶电泳法和液相色谱-串联质谱仪方法兼容。

 DESCRIPTION (provided by applicant): In this proposal we seek to demonstrate a new experimental approach to develop a new subtractive method to remove abundant proteins from complex human fluid samples. The ultimate goal is to develop a suite of methods or tools to facilitate the de novo discovery of a complete set of low-abundance potential protein biomarkers from which more targeted studies can be performed. A particular interest is in the discovery of potential biomarkers for cancers, and in the past we have studied lung cancer samples and references drawn from the same cohort. In this Phase I proposal, we seek to demonstrate the selective and specific removal of human serum albumin isoforms and dimers from human plasma. These serum albumins comprise half of the protein in human serum or plasma, and cause very real issues with both tandem mass spectrometry and difference gel electrophoresis approaches. The Phase II would extend to work to remove three orders of magnitude of abundant proteins, a 50 times increase from existing state-of-the-art methods. One key feature is that the conditions are chosen to minimize protein-protein interactions, which compromise the performance of existing methods. The simplified human fluid samples produced will be compatible with both 2D gel electrophoresis and liquid chromatography- tandem mass spectrometry approaches.