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Molecular & Cellular Analysis Technologies
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The advent of high density DNA microarrays almost 25 years ago has been an essential step in the genomics revolution. Major successes include genome-wide gene expression profiling (GEP) which has led to a new understanding of cellular control pathways and powerful diagnostic/prognostic tests such as for predicting breast cancer recurrence. In principle, a similar approach, namely the utilization of high density protein microarrays, could herald a revolution in proteomics, particularly in the field of cancer research.Potential applications include measuring protein-protein interactions, immune response profiling aimed at discovery of tumor autoantibody biomarkers and profiling enzyme substrate interactions involved in signal transduction (e.g. kinase-substrate interactions). However, despite continued progress, high-density protein microarrays have not yet lived up to their original promise. Current limitations include low array density, poor reproducibility, high cost, poor assay kinetics and difficulty in detecting a diversity of bait-prey interactions including enzyme-induced protein modifications. In contrast, mass spectrometry used in conventional proteomics provides many important capabilities including protein identification by sequencing (and fingerprinting) as well as molecular-level identification of protein modifications (e.g. phosphorylation), yet does not have the parallelization and high throughput capacity of microarrays. During Phase I we evaluated the feasibility of a new proteomic approach developed by AmberGen termed Bead- based Global Proteomic Screening (Bead-GPS'). This method combines the advantages of mass spectrometry (MS) and microarray technology by using correlated MALDI mass spectrometric imaging (MALDI-MSI) and fluorescence imaging of photocleavable Mass-Tag-coded random bead-arrays. All of the specific aims were achieved including: i) Demonstration of Bead-GPS' synchronized fluorescent and MALDI-MS imaging; ii) Demonstration of high capacity coding of protein/peptide bead libraries; iii) Development of improved photocleavable linkers; iv) Development of improved micro-well substrates for the bead-arrays; v) Demonstration of the feasibility to use Bead-GPS' to screen large combinatorial synthesized one-bead/one-compound (OBOC) bead-libraries comprised of photocleavable peptides/biomimetic peptides. During Phase II, we will further optimize major components of Bead-GPS' including the construction of photocleavable bead-libraries of peptides and peptoids, formatting the libraries into high density random bead- arrays and obtaining correlated fluorescence and MALDI-MS images of the bead-arrays to identify and decode bio- molecular interactions of 'prey' molecules with the bead-library (the 'bait'). Specific emphasis will be on validating Bead-GPS' for two applications relevant for cancer research and clinical diagnosis. The first application involves use of Bead-GPS' to screen mid-size, parallel synthesized, photocleavable peptide bead-libraries and then larger combinatorial synthesized OBOC libraries for kinase substrate interactions. The second application is the use of Bead-GPS' to screen large combinatorial OBOC biomimetic peptide libraries (peptoids) against patient sera for the discovery of surrogate antigens that can be used as biomarkers in cancer immunoassays. In order to accelerate the development and commercialization of Bead-GPS', AmberGen will work a closely with Dr. Ron Zuckermann, Director of the Biological Nanostructure Facility at the Molecular Foundry located at Berkeley and a recognized pioneer in the field of biomimetic peptide synthesis; Dr. Cathy Costello, Director, Center for Biomedical Mass Spectrometry at Boston University Medical School and a leading expert in the field of MALDI and its biomedical applications; Dr. Gary Kruppa, V.P. of Business Development at Bruker Daltonics (Billerica, MA), a world-leading provider of MALDI-MS instrumentation; and Dr. Paul Billings, the Chief-Medical Officer at Life Technologies, a leading company in marketing biotechnology products and services.