Affinity-based Mass Spectrometry Protein Assays

描述（由申请人提供）： 本阶段I-阶段II快速通道研究提案的目标是推进质谱免疫测定（MSIA）-生物反应性质谱仪探针（BRP）组合方法的开发，以实现强大的多路复用技术，最终将用于人群筛查应用和癌症研究相关研究。MSIA和BRP技术分别由Intrinsic Bioprobes Inc.开发。一直都是这样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用于随后的MALDI-TOF MS分析。当在分析中引入内标物时，可以实现测定蛋白质的绝对和/或相对定量。另一方面，生物反应性探针通过利用能够消化微量蛋白质的MALDI靶向表面固定化酶，帮助进行彻底的蛋白质结构表征。当结合在一起时，这两种技术可以产生一种复杂的方法，可以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I期研究中，我们建议通过使用几种模型蛋白和MSIA测定法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组合方法的鲁棒性和高通量能力。我们还将继续开发用于血浆和尿液中内源性蛋白质的额外MSIA-BRP检测方法，其中一些已被指示为潜在的癌症生物标志物。许多这些蛋白质和检测将用于开发和评价一种新的，相对定量MSIA方法。最后，将在大规模环境（200 - 300份样本，尿液和血浆）中测试I期和II期设计的检测试剂盒和方案，以验证MSIA-BRP技术用于人群筛查实验的潜力。该I期-II期研究的最终产品将是MSIA-BRP平台和检测试剂盒的组合，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。

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.