Affinity-based Mass Spectrometry Protein Assays

基于亲和力的质谱蛋白质检测

• 批准号: 6787452
• 负责人: DOBRIN NEDELKOV
• 金额: $ 89.07万
• 依托单位: INTRINSIC BIOPROBES, INC.
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-03-24 至 2005-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6787452
• 关键词: affinity labeling; blood; clinical research; human tissue; immunologic assay /test; mass spectrometry; matrix assisted laser desorption ionization; nucleic acid probes; technology /technique development; urine

DESCRIPTION (provided by applicant): The objective of this Phase I - Phase II Fast Track Research Proposal is to advance the development of a combined Mass Spectrometric Immunoassay (MSIA) - Bioreactive Mass Spectrometer Probes (BRP) approach to a robust multiplexed technology that will ultimately be used in population screening applications and in studies relevant to cancer research. Individually, the MSIA and BRP technologies had been under development at Intrinsic Bioprobes Inc. for the last several years. Each technology brings a unique perspective to protein analysis. The MSIA approach utilizes affinity captures to selectively retrieve proteins from biological samples for subsequent MALDI-TOF MS analysis. When internal standards are incorporated in the analysis, absolute and/or relative quantitation of the assayed proteins can be achieved. On the other hand, the Bioreactive Probes aid in the thorough structural protein characterization via utilization of MALDI target surface-immobilized enzymes capable of digesting minute amounts of proteins. When combined together, the two technologies can yield a complexed approach that enables sensitive detection, quantitation and structural analysis of specific proteins from complex biological samples. In the Phase I Research, we propose to evaluate and demonstrate the reproducibility and sensitivity of the MSIA technology via execution of well-designed experiments using several model proteins and MSIA assays. In Phase II, we will demonstrate the robustness and the high-throughput capability of the combined MSIA-BRP approach. We will also proceed with the development of additional MSIA-BRP assays for proteins endogenously present in plasma and urine, some of which have been indicated as potential cancer biomarkers. A number of these proteins and assays will be utilized for the development and evaluation of a novel, relative quantitation MSIA approach. And finally, the assays and protocols designed in the Phase I and Phase II will be tested in a large-scale setting (200-300 samples, both urine and plasma) to validate the potential of the MSIA-BRP technology for population screening experiments. The ultimate product resulting from this Phase I-Phase II Research will be a combined MSIA-BRP platform and assays capable of assessing both qualitative and quantitative modulations in proteins analyzed directly from plasma, urine, or other biological fluid or tissue.

描述（由申请人提供）： 该第一阶段 - 第二阶段快速通道研究提案的目标是推进质谱免疫分析 (MSIA) - 生物反应质谱探针 (BRP) 组合方法的开发，以实现强大的多重技术，最终将用于人群筛查应用和与癌症研究相关的研究。过去几年，Intrinsic Bioprobes Inc. 一直在单独开发 MSIA 和 BRP 技术。每种技术都为蛋白质分析带来了独特的视角。 MSIA 方法利用亲和捕获从生物样品中选择性地检索蛋白质，以进行后续的 MALDI-TOF MS 分析。当分析中加入内标时，可以实现被测蛋白质的绝对和/或相对定量。另一方面，生物反应探针通过利用能够消化微量蛋白质的 MALDI 目标表面固定化酶，有助于彻底的结构蛋白质表征。当结合在一起时，这两种技术可以产生一种复杂的方法，能够对复杂生物样品中的特定蛋白质进行灵敏的检测、定量和结构分析。在第一阶段研究中，我们建议通过使用多种模型蛋白和 MSIA 检测执行精心设计的实验来评估和证明 MSIA 技术的重现性和灵敏度。在第二阶段，我们将展示 MSIA-BRP 组合方法的稳健性和高吞吐量能力。我们还将继续开发针对血浆和尿液中内源性蛋白质的其他 MSIA-BRP 检测方法，其中一些已被表明是潜在的癌症生物标志物。其中许多蛋白质和检测方法将用于开发和评估新型相对定量 MSIA 方法。最后，第一阶段和第二阶段设计的检测方法和方案将在大规模环境（200-300 个样本，尿液和血浆）中进行测试，以验证 MSIA-BRP 技术用于人群筛查实验的潜力。第一阶段和第二阶段研究的最终产品将是一个组合的 MSIA-BRP 平台和检测方法，能够评估直接从血浆、尿液或其他生物液体或组织中分析的蛋白质的定性和定量调节。