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

描述（由申请方提供）：本提案的目的是引入一种产品，该产品将使通过凝胶电泳（SDS-PAGE）进行的蛋白质分离能够以高效、高通量的方式通过质谱法作为肽进行分析。这种设备将为生物学和医学研究人员提供一种引人注目的新方法，用于分析复杂的蛋白质组学样品（100 - 1000个单个组分），直接解决蛋白质组学研究中最持久的挑战;如何管理样品组分的多样性，在数量和浓度方面，以最大限度地提高样品物种检测，同时保留与系统生物学相关的信息。由此产生的产品可用性 通过将最有效、最高分辨率的蛋白质分离技术与肽的质谱检测的准确性、灵敏度和速度以彻底的方式结合起来，工作将解决这些挑战。目前，这种联系并不存在。蛋白质或蛋白质家族变异的细微细节与癌症、阿尔茨海默病、糖尿病和关节炎等威胁性疾病的检测或传播有关。因此，实验协议，使这些功能的识别和识别是必要的，以满足研究目标。这种新产品将为蛋白质组学研究工作提供一个有吸引力的实验方案，因为样品分离将在完整蛋白质的水平上进行，保留了有关给定物种内多态性和翻译后修饰的有价值信息。本第1阶段研究的目标是证明一种用于将蛋白质凝胶分离与肽的质谱检测耦合的系统，该系统简单、高效、灵敏、可重现且易于实施。第一阶段可行性研究列出了四个具体目标，并就如何测试和评估这些目标的成功进行了后续讨论。 自从Laemilli在大约40年前引入以来，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蛋白质分离的几乎普遍适用性、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。其原因包括肽检测的准确性、灵敏度和速度的关键分析特性，以及选择性片段化肽以获得使蛋白质鉴定成为可能的一级序列信息的能力。不幸的是，目前，凝胶之间没有有效和彻底的过渡， 蛋白质的分离和肽的质谱分析。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，并且通常不完整和不精确。概述和初步证明，在这个建议是一种新的膜，有能力捕获和消化蛋白质（S）正交印迹从SDS-PAGE凝胶。膜中构建的化学物质的特定选择和组成用于就地处理蛋白质，将其彻底消化成其组成肽，同时保留和限制扩散，直到通过应用适当的洗脱溶剂将其有意释放。据信，由该膜执行的双重任务可以以及时和精确的方式发生，使其成为非常可行的商业产品。如所设想的，完成该系统所需要的是用于从膜上洗脱结合的肽的手段，使得可以一次处理包括整个凝胶的分离。该程序的概念是创建洗脱板， 洗脱溶剂在离心力的作用下穿过膜。这些洗脱板的设计将允许洗脱溶剂和分析物容易地收集，同时保持分离的分辨率。这个产品的目标是这些。1)利用凝胶蛋白质分离的普遍适用性、便利性和分辨能力，分离和保留复杂蛋白质混合物的一级结构。2)利用电泳印迹的效率和方便性，从分离凝胶中彻底去除所有蛋白质，而无需检测。3)在保持分离分辨率的前提下，将凝胶分离的蛋白质彻底消化成组成肽。4)捕获并浓缩所得肽，并将其在优化用于质谱检测的液体介质中进行包埋。5)利用质谱法检测多肽的准确性、灵敏度和速度来询问样品，以实现研究目标。该产品的关键组成部分将是独特设计的双功能膜和新型洗脱板，这将使酶消化的蛋白质（肽）在与质谱法检测兼容的溶液中进行洗脱。

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.