Development of SPR/MS protein array platform

这项建议的主要技术目标是发展表面等离子体共振质谱学 利用表面等离子体共振(SPR)和MALDI-TOF质量的(SPR/MS)蛋白质阵列平台 用于检测蛋白质和描绘蛋白质-蛋白质相互作用的光谱。在第一个可行性/试点中 阶段，我们将检查蛋白质排列到SPR活性表面，亲和检索蛋白质上的 阵列表面，并用MALDI-TOF MS读出蛋白质相互作用。功能活性蛋白质阵列将 通过将抗体和蛋白质斑点(排列)和固定到芯片表面而产生。这个 蛋白质阵列将用于从溶液中亲和检索蛋白质，之后阵列将被 通过MALDI-TOF质谱仪进行分析，以评估MS读数的可行性 亲和力-从蛋白质阵列上的斑点捕获蛋白质。在圆满完成这些任务后， 我们将进入第二个扩展开发阶段，在这个阶段，高分辨率SPR检测将 和集成的SPR/MS蛋白质阵列平台将用于检测蛋白质和 来自生物流体的蛋白质-蛋白质相互作用。将使用高分辨率SPR阵列仪器来 显示对阵列上的蛋白质相互作用(从单个点)进行量化的可行性。这个 SPR/MS蛋白质阵列平台的组件与实验对照和 变量，将进一步发展和优化。如果需要，更高性能的微阵列将 以及SPR/MS蛋白质阵列平台在检测蛋白质和 将评估来自血浆和尿液等生物液的蛋白质-蛋白质相互作用。最终结果 这一发展研究将是一个蛋白质芯片平台和方法，可以应用于各种 蛋白质组学研究系列，包括高通量生物标志物分析、蛋白质-蛋白质相互作用、 人口筛选工作，蛋白质治疗监测，疾病机制结构的探索， 和诊断化验的发展。最终，SPR/MS蛋白质阵列平台可以实现快速、 并行、高通量筛选蛋白质生物标志物，使用通过最低限度获得的样本 侵入性样本采集方法，将筛查工作推广到临床和诊断 实验室。

The main technical objective of this proposal is to develop Surface Plasmon Resonance Mass Spectrometry (SPR/MS) protein array platform that utilizes Surface Plasmon Resonance (SPR) and MALDI-TOF mass spectrometry for detection of proteins and delineation of protein-protein interactions. In the first feasibility/pilot phase, we will examine the protein arraying to an SPR-active surface, affinity retrieval of proteins on the array surface, and MALDI-TOF MS readout of the protein interactions. Functionally-active protein array will be created via spotting (arraying) and immobilization of antibodies and proteins onto a chip surface. The protein array will then be used for affinity-retrieval of proteins from solution, after which the array will be analyzed via MALDI-TOF mass spectrometry to gauge the feasibility of the MS readout of the affinity-captured proteins from the spots on the protein array. Upon the successful completion of these tasks, we will move into the second, expanded development phase, where high-resolution SPR detection will be incorporated and the integrated SPR/MS protein array platform will be used for detection of proteins and protein-protein interactions from biological fluids. A high resolution SPR array instrument will be employed to show the feasibility of quantification of the protein interactions (from individual spots) on the array. The interface between the components of the SPR/MS protein array platform, and the experiment controls and variables, will be further developed and optimized. If needed, a higher-performance microarrayer will be incorporated, and the performance of the SPR/MS protein array platform in detection of proteins and protein-protein interactions from biological fluids such as plasma and urine will be evaluated. The final result of this developmental research will be a protein chip platform and methods that can be employed into various lines of proteomics research, including high-throughput biomarker analysis, protein-protein interactions, population screening efforts, therapeutic monitoring of proteins, exploration of disease mechanism structures, and diagnostic assays development. Ultimately, the SPR/MS protein array platform could enable rapid, parallel, and high-throughput screening of protein biomarkers, using samples obtained through minimally invasive sample collection methods, propagating the screening efforts into the clinical and diagnostic laboratories.