The overall objective of this project is to develop a cancer biomarker discovery and validation platform for analyses e.g. of human blood plasma and other clinically relevant samples that will provide measurements that are:much more robust, of higher sensitivity, provide more than order of magnitude throughput, and have improved quantitative utility compared to existing platforms. The new platform will provide increased dynamic range, and thus proteome coverage, and allow quantitative measurements of candidate cancer biomarker proteins at concentrations that are presently problematic for broad proteome measurements (e.g.plasma protein concentrations similar to PSA and lower). The enhanced throughput of the platform will be realized in conjunction with improved quality of quantitative data (derived from increased robustness, higher run-to-run reproducibility, and increased sensitivity), providing greater confidence in proteomic data. Thus, the new platform will be a powerful tool for both candidate biomarker discovery and pre-clinical candidate validation, and with further refinement provide the basis for broad clinical application. The instrumental platform will be an integration of fast liquid chromatography (LC)based separations, a novel microfabricated array nanoelectrospray ionization (nanoESI) interface, and high resolution gas phase ion mobility separations coupled with accurate mass time-of-flight mass spectrometry. In the initial R21 phase, we will focus primarily on: (1) the development of a prototype instrument platform and the key nanoESI interface component; and (2) an initial evaluation of the platform's throughput, sensitivity, and robustness. The R33 Phase will focus on: (1) the further refinement of the platform, (2) the development of the informatics tools needed to support the platform's massive data production rates, and (3) the platform's evaluation in the context of high throughput quantitative proteomics measurements involving clinically relevant oncology-based human blood plasma samples and the comparison with existing assays for a number of low level protein biomarkers (e.g.PSA). The anticipated result of this project will be a robust high throughput platform with the potential for revolutionizing cancer biomarker discovery and validation, and its initial dissemination for clinical applications.