Sample Pretreatment for Human Fluids

描述(申请人提供)：在这项提案中，我们寻求开发一种新的方法和仪器，以降低从人体体液中提取的蛋白质混合物的复杂性，着眼于发现非常低丰度的蛋白质生物标记物。该方法完成两个任务：它将蛋白质混合物的动态范围减小三个或更多个数量级，并将蛋白质浓缩100倍或更多，以便能够检测到低丰度并注意到浓度差异。该方法不使用抗体、组合多肽库、载体两性离子或固-液吸附平衡，如高效液相色谱法。所制得的样品与二维凝胶电泳法和二维液相色谱串联质谱方法兼容。通过使用不同的物理方法来分离蛋白质混合物，目标是发现现有最先进方法无法实现的新生物标记物。开发完成后，建议的方法和仪器将使用人类血浆进行演示，其中来自肺癌患者的样本将与未患癌症的参照物进行比较。与传统的免疫耗竭方法相比，将发现额外的低丰度蛋白质，这些蛋白质是潜在的生物标志物。

DESCRIPTION (provided by applicant): In this proposal we seek to develop a new method and instrument to reduce the complexity of protein mixtures derived from human fluids with an eye to discovery of very low-abundance protein biomarkers. The proposed method accomplishes two tasks: it reduces the dynamic range of the protein mixture by three or more orders of magnitude and it concentrates proteins by a factor of 100 or more so that low- abundance can be detected and concentration differences noted. The method does not use antibodies, combinatorial peptide libraries, carrier ampholytes or solid-liquid adsorption equilibria such as HPLC. The samples produced are compatible with 2D gel electrophoresis and 2D liquid chromatography tandem mass spectrometry approaches. By using different physical methods to separate the protein mixtures, the goal is to uncover new biomarkers that have eluded existing state-of-the-art methods. After development, the proposed method and instrument will be demonstrated using human plasma, where a sample from a lung cancer patient is compared with a reference that does not have cancer. Additional low-abundance proteins that are potential biomarkers will be uncovered when compared with traditional immunodepletion methods.