Novel membrane to link SDS-PAGE with mass spectrometry for proteomic studies of d

描述(由申请人提供)：本提案的目标是介绍一种产品，该产品将能够以高效、高通量的方式通过凝胶电泳法(SDS-PAGE)将蛋白质分离为多肽，并通过质谱仪进行分析。这种设备将为生物和医学研究人员提供一种引人注目的新方法，通过直接解决蛋白质组研究中最持久的挑战，分析复杂的蛋白质组样本(100-1000个单独成分)；如何在数量和浓度上管理样本成分多样性，以最大限度地检测样本物种，同时保留与系统生物学相关的信息。由此产生的产品的可用性 这项工作将通过将最高效、最高分辨率的蛋白质分离技术与以彻底方式进行的多肽的准确、灵敏和快速的质谱学检测相结合来解决这些挑战。目前，这种联系并不存在。蛋白质或蛋白质家族变异的微妙细节与癌症、阿尔茨海默氏症、糖尿病和关节炎等危险疾病的检测或传播有关。因此，能够识别和识别这些特征的实验方案对于满足研究目标是必要的。这一新产品将为蛋白质组研究工作提供一个有吸引力的实验方案，因为样品将在完整蛋白质水平上进行分离，保留有关给定物种内的多态和翻译后修饰的有价值的信息。这项第一阶段研究的目标是展示一种简单、高效、灵敏、可重复性和易于实施的将蛋白质凝胶分离与多肽的质谱学检测相结合的系统。列出了第一阶段可行性研究的四个具体目标，并就如何测试和评估它们的成功进行了后续讨论。自40年前Laemilli提出以来，SDS-PAGE一直是分离和分析蛋白质和蛋白质混合物的最流行的方法之一。SDS-PAGE分离蛋白质的近乎普遍的适用性、分辨率、一致性和简单性仍然是无与伦比的。在过去的15年里，质谱仪已经成为实验蛋白质组学中的首选检测器。其原因包括检测多肽的准确性、灵敏度和速度等关键的分析特性，以及选择性地对多肽进行片断以获得使蛋白质鉴定成为可能的初级序列信息的能力。不幸的是，目前还没有有效和彻底的凝胶之间的过渡 蛋白质的分离和多肽的质谱分析。目前试图将这两种强大的实验方法联系起来的协议主要是手动的、耗时的，而且往往不完整和不精确。该方案概述并初步证明了一种新型膜，它具有捕获和消化从十二烷基硫酸钠-PAGE凝胶中正交印迹的蛋白质(S)的能力。膜内化学成分的特定选择和组成可就地处理蛋白质(S)，将它们彻底消化成组成它们的多肽，同时保留和限制扩散，直到它们通过应用适当的洗脱溶剂故意释放。人们相信，这种膜所执行的对决任务可以及时和精确地进行，使其成为一种极其可行的商业产品。正如设想的那样，完成该系统所需要的是一种从膜上集体洗脱结合多肽的方法，以便可以一次处理围绕整个凝胶的分离。这一过程的概念是创建洗脱板，以使 洗脱溶剂在离心力作用下通过膜。这些洗脱板的设计将允许在保留分离分辨率的情况下轻松收集洗脱溶剂和分析物。这款产品的目标是这些。1)利用基于凝胶的蛋白质分离的近乎普遍的适用性、方便性和分辨率来分离和保存复杂蛋白质混合物的一级结构。2)利用电泳法的效率和方便性，在不需要检测的情况下，将所有蛋白质从分辨凝胶中彻底去除。3)在保持分离分辨率的同时，将凝胶分离的蛋白质完全消化成其组成的多肽。4)捕获和浓缩所得的多肽，并将它们洗脱在优化的液体介质中，以便通过质谱学进行检测。5)利用多肽的准确、灵敏和快速的质谱学检测来询问样品，以达到研究的目的。该产品的关键组件将是独特设计的双功能膜和新型洗脱板，该洗脱板将使酶消化的蛋白质(肽)能够在与质谱学检测兼容的溶液中进行集体洗脱。

DESCRIPTION (provided by applicant): The objective of this proposal is to introduce a product that will enable protein separations by gel electrophoresis (SDS-PAGE) to be analyzed as peptides by mass spectrometry in an efficient, high-throughput manner. Such a device will offer investigators in biology and medicine a compelling new method for analyzing complex proteomic samples (100s - 1000s of individual components) by directly addressing the most persistent challenges in proteomic research; how to manage sample component diversity, in both number and concentration, to maximize sample species detection while preserving information pertinent to the biology of the system. Availability of the product resulting from this work will address these challenges by coupling the most efficient, highest resolution, protein separation technique with the accuracy, sensitivity and speed of mass spectrometry detection of peptides in a manner that is thorough. Presently, this link does not exist. Subtle details in the variants of a protein or protein family are implicated in the detection or propagation of menacing diseases like cancer, alzheimier's, diabetes and arthritis. Therefore, experimental protocols that enable the discernment and identification of these features are necessary to meet research objectives. This new product will offer an attractive experimental scheme to proteomic research efforts because sample separation will be performed at the level of intact proteins, preserving valuable information regarding polymorphisms and post- translational modifications within given species. The goal of this phase 1 study is to demonstrate a system for coupling protein gel separations with mass spectrometry detection of peptides that is simple, efficient, sensitive, reproducible and easily implemented. Four specific aims for this phase 1, feasibility study are listed with follow-up discussion on how they will be tested and evaluated for success. Since its introduction by Laemilli ~ 40 years ago, SDS-PAGE has remained one of the most popular methods for separating and analyzing proteins and protein mixtures. The near universal applicability, resolving power, consistency and simplicity of SDS-PAGE protein separation remains unmatched. Over the past ~15 years, the mass spectrometer has emerged as the detector of choice in experimental proteomics. The reasons for this include the critical, analytical-traits of accuracy, sensitivity and speed in detection of peptides and the ability to selectively fragment peptides to obtain primary sequence information enabling protein identification. Unfortunately, currently, there is no efficient and thorough transition between gel separation of proteins and mass spectrometry of peptides. Current protocol that attempts to link these two powerful experimental methods is largely manual, time consuming and often incomplete and imprecise. Outlined and preliminarily demonstrated in this proposal is a novel membrane that has the ability to both capture and digest protein(s) orthogonally blotted from SDS-PAGE gels. Specific selection and composition of the chemistry built into the membrane acts to process protein(s) in-place, thoroughly digesting them into their constituent peptides while retaining and limiting diffusion, until their deliberate release by application of the appropriate elution solvent. It is believed that the duel tasks performed by this membrane can take place in a timely and precise manner making it an extremely viable commercial product. What is needed to complete the system, as envisioned, is a means for en-masse elution of bound peptides from membrane so that separations encompassing entire gels can be processed at once. The concept for this procedure is the creation of elution plates that will allow elution solvent to pass through the membranes under the force of centrifugation. The design of these elution plates will allow the elution solvent and analyte to be easily collected with the resolution of separation preserved. The goals for this product are these. 1) To harness the near universal applicability, convenience, and resolving power of gel-based protein separations to separate and preserve the primary structure of complex protein mixtures. 2) To harness the efficiency and convenience of electrophoresis blotting to thoroughly remove all proteins from the resolving gel without the need of detection. 3) To thoroughly digest the gel-separated proteins into their constitute peptides while maintaining the resolution of separation. 4) To capture and concentrate the resulting peptides and elute them in a liquid medium optimized for detection by mass spectrometry. 5) Use the accuracy, sensitivity and speed of mass spectrometry detection of peptides to interrogate the sample to fulfill the objectives of the research. The key components of the product will be the uniquely designed bi-functional membrane and the novel elution plate that will enable the en-masse elution of the enzyme-digested proteins (peptides) in a solution compatible with detection by mass spectrometry.