Sequencing of the human genome has led to a new and even more ambitious goal - characterization of the human proteome. Such an endeavor involves not only understanding the function of hundreds of thousands of different proteins expressed in human cells but also characterizing the millions of potential interactions that can occur with other cellular and extracellular molecules including proteins, nucleic acids, lipids and small molecules. The ability to rapidly perform such massive global proteomic screens would be a powerful tool in many areas of cancer research such as biomarker discovery, mapping cellular networks and drug development. Although high-density DNA microarrays introduced almost 20 years ago have had a major impact in facilitating the genomic revolution, high-density protein microarrays have not yet exerted a similar impact. Current limitations in protein microarray technology include low array density, poor reproducibility, high cost, poor assay kinetics and difficulty in detecting a diversity of bait-prey interactions as well as enzyme-induced protein modifications. In contrast, mass spectrometry used in conventional proteomics does provide many of these capabilities including label-free identification of small drug compounds, identification of protein modifications and protein identification. However, the separation methods used in conjunction with conventional mass spectrometry based proteomics such as two-dimensional gel electrophoresis and liquid chromatography are slow and not nearly as robust as the physical arraying/sorting of proteins inherent in a microarray. During Phase I we will evaluate a new approach developed by AmberGen for proteomics termed Bead-based Global Proteomic Screening (Bead-GPSTM) which combines the advantages of MALDI mass spectrometry imaging (MALDI-MSI) and microarray technology. This approach utilizes photocleavable mass-tags (PC-Mass-Tags) to encode a protein-bead library (bait library) as well as interacting prey molecules such as other proteins, all displayed on individual beads randomly arrayed at high-density (1,000,000 wells) in a Pico-well plate. Because we have shown in preliminary experiments that MALDI-MSI of high density protein-bead arrays has the potential to rapidly identify millions of different mass-tag combinations, with high sensitivity and spatial resolution, it is possible to perform highly multiplexed screening of bait-prey interactions far beyond the capabilities of conventional fluorescence microarrays. However, fluorescence imaging can still be used with Bead-GPS" to pre- identify and quantitate positive interactions which are then decoded by MALDI-MSI. In addition, the power of Bead-GPSTM is further extended by the ability of MALDI-MSI to perform on-bead label-free detection of i) interacting prey molecules such as small drug compounds, ii) other proteins (protein fragmentation fingerprinting) and iii) protein modifications (e.g. serine or tyrosine phosphorylation). During Phase I we will fabricate a 100-member prototype protein-bead library using cell-free protein translation techniques in order to evaluate key features of Bead-GPSTM including PC-Mass-Tag coding (for both bait and prey molecules), protein-protein interaction analysis both with PC-Mass-Tags and by label-free means, detection of label-free protein-drug interactions, detection of protein modifications and serum profiling for cancer biomarker discovery. During Phase II, a full proteome-wide Bead-GPS" platform will be constructed and tested. In order to accelerate commercialization of the products resulting from this project we will work closely during Phase I and II with Bruker Daltonics (Billerica, MA), a world-leading provider of MALDI-MS instrumentation, to develop a user- friendly, fully integrated instrument (and software) which will serve as a platform for the Bead-GPS" technology. PUBLIC HEALTH RELEVANCE: Although high density DNA microarrays introduced almost 20 years ago have had a major impact in facilitating the genomic revolution, a similar impact has not yet occurred in the field of proteomics despite the availability of high density commercial protein microarrays. We will evaluate in Phase I a new approach for proteomics termed Bead- based Global Proteomic Screening (Bead-GPSTM) which overcomes existing limitations in proteomic technology by combining the advantages of MALDI mass spectrometric imaging and microarrays. Potential benefits of the new approach include the discovery of new biomarkers for cancer diagnostics, increased understanding of the causes of cancer and discovery of new drugs to treat cancer.