Our research is focused on developing integrated systems that provide analytical tools needed for truly global analyses of biological systems. The approach employs multi-dimensional separations coupled to high- performance MALDI TOF mass spectrometers via new three-dimensional sample plates using collimated hole structures (CHS) and monolithic supports as an alternative to established LC-MS-MS systems employing electrospray ionization. Recent advances in high performance ion trap mass spectrometers employing electrospray ionization and nanoflow liquid chromatography have significantly improved capabilities for analyzing biological samples, but this technology has serious inherent limitations on dynamic range and speed. Early applications of MALDI were limited by relatively low resolving power and mass accuracy of available TOF instruments, and the lack of a commonly available interface with liquid chromatography. Our recent work has overcome these limitations and high performance MALDI-TOF and TOF-TOF instruments interfaced with high capacity separations are now available. These instruments provide speed, resolution, dynamic range, and sensitivity orders of magnitude superior to the performance of commercially available instruments. These instruments and interfaces provide the performance required for global analysis of biological samples, but the database and bioinformatics tools proposed for development in this project are essential for converting this massive volume of data to useful information. Although global proteomics is clearly much more challenging than sequencing the human genome, the technology developed in this and related projects will provide the tools required for significant progress in the proteomics analog to the human genome project. The major remaining challenge involves converting the massive volume of data than can be produced by these instruments into information that can be readily used to address biological problems. This is the focus of the proposed project. PUBLIC HEALTH RELEVANCE: Major initiatives by The Human Proteome Organization are focused on plasma, liver, and brain proteomics. The premise of these initiatives is that "A comprehensive, systematic characterization of circulating proteins in health and disease will greatly facilitate development of biomarkers for prevention, diagnosis, and therapy of cancer and other diseases." This project will demonstrate that the technology described in this proposal provides a feasible route to achieving the goals of these initiatives.