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

将 SDS-PAGE 与质谱联用的新型膜用于 d 的蛋白质组学研究

• 批准号: 8714346
• 负责人: Stephen Hattan
• 金额: $ 13.04万
• 依托单位: VIRGIN INSTRUMENTS CORPORATION
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-05-05 至 2014-11-04
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8714346
• 关键词: 3-Dimensional; Address; Area; Arthritis; Binding; Biology; Cells; Centrifugation; Chemistry; Complex; Coupling; Detection; Devices; Diabetes Mellitus; Diagnosis; Diffusion; Digestion; Disease; Electrophoresis; Enzymes; Escherichia coli; Evaluation; Feasibility Studies; Gel; Genetic Polymorphism; Goals; Individual; Link; Liquid substance; Logic; Malignant Neoplasms; Manuals; Mass Spectrum Analysis; Measurable; Measures; Medicine; Membrane; Methods; Metric; Parents; Peptide Fragments; Peptide Signal Sequences; Peptides; Performance; Phase; Plastics; Post-Translational Protein Processing; Procedures; Process; Protein Family; Proteins; Proteomics; Protocols documentation; Reporting; Reproducibility; Research; Research Personnel; Resolution; Sampling; Scheme; Signal Transduction; Site; Slice; Solutions; Solvents; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Speed; Staining method; Stains; Structure; System; Systems Biology; Techniques; Technology; Testing; Time; Variant; Work; Yeasts; base; design; detector; follow-up; gel electrophoresis; high throughput analysis; mass spectrometer; meetings; novel; phase 1 study; polyvinylidene fluoride; protein complex; public health relevance; success; trait

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.

描述（由申请人提供）：本提案的目的是介绍一种产品，该产品能够通过凝胶电泳（SDS-PAGE）分离蛋白质，并通过质谱法以有效、高通量的方式将蛋白质分析为肽。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，通过直接解决蛋白质组研究中最持久的挑战来分析复杂的蛋白质组样本（数百至数千个单独的成分）；如何管理样品成分的数量和浓度多样性，以最大限度地检测样品种类，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 工作将通过将最有效、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来解决这些挑战。目前，此链接不存在。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，能够辨别和识别这些特征的实验方案对于实现研究目标是必要的。该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，保留有关给定物种内多态性和翻译后修饰的有价值的信息。该第一阶段研究的目标是展示一种将蛋白质凝胶分离与肽质谱检测相结合的系统，该系统简单、高效、灵敏、可重复且易于实施。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是否成功进行了后续讨论。 自 Laemilli 约 40 年前推出以来，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 蛋白质分离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。在过去约 15 年里，质谱仪已成为实验蛋白质组学的首选检测器。其原因包括肽检测的准确性、灵敏度和速度等关键分析特性，以及选择性片段化肽以获得初级序列信息以实现蛋白质鉴定的能力。不幸的是，目前，凝胶之间还没有有效、彻底的过渡。 蛋白质的分离和肽的质谱分析。当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且不精确。该提案概述并初步证明了一种新型膜，它能够捕获和消化从 SDS-PAGE 凝胶上正交印迹的蛋白质。膜中化学物质的具体选择和组成可就地处理蛋白质，将它们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有意释放它们。人们相信，这种膜所执行的双重任务可以及时、精确地完成，使其成为一种极其可行的商业产品。正如所设想的，完成该系统所需的是一种从膜上整体洗脱结合肽的方法，以便可以立即处理涵盖整个凝胶的分离。该程序的概念是创建洗脱板，允许 洗脱溶剂在离心力的作用下透过膜。这些洗脱板的设计将允许轻松收集洗脱溶剂和分析物，同时保留分离分辨率。该产品的目标如下。 1) 利用基于凝胶的蛋白质分离的近乎普遍的适用性、便利性和分辨率来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便利性，无需检测即可彻底去除分离胶中的所有蛋白质。 3) 将凝胶分离的蛋白质彻底消化成其组成肽，同时保持分离的分辨率。 4) 捕获并浓缩所得肽，并在针对质谱检测而优化的液体介质中将其洗脱。 5) 利用质谱检测肽的准确性、灵敏度和速度来询问样品以实现研究目标。该产品的关键部件将是独特设计的双功能膜和新型洗脱板，能够在与质谱检测兼容的溶液中对酶消化的蛋白质（肽）进行整体洗脱。