? 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. ? ?
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