This Proteome & Genome Core (PPG Core B) will provide essential, cutting edge, proteomic/transcriptomic/genetic expertise to facilitate the translational impact of this highly integrated PPG. This PPG Core will be responsible for providing the research investigators with a well characterized ARDS biobank (i.e. DNA from European descent and African descent subjects), a complete list of tagging single nucleotide polymorphisms (tSNPs) for each PPG candidate gene, mid-throughput genotyping services, and data analysis tools to test for association between PPG-studied SNPs and susceptibility and severity of ARDS. The functional non-synonymous coding SNPs will be screened in silico to identify their impact on protein structure and function (protein-protein interaction, catalytic activity, post-translational modification) by computational protein structure molecular modeling, with protein-protein interaction validated by surface plasmon resonance (SPR) screening. Effects of exon-intron boundary SNPs on alternative splicing of these key genes will be analyzed by in silico prediction and further validated by SNP-containing minigene constructs in individual projects. PPG Core B will provide the service to generate the mutated DNA constructs (with disease-associated SNP) to generate recombinant protein used in SPR, or GRF-labeled protein for cellular location tracking in endothelial cells (to be used in Core D and individual projects). In addition, this PPG Core will further validate the genomic expression data by proteomic approaches (2-D protein gel and mass spectrometry). On top of the protein level characterization, the PPG Core B will define the protein translational modification (PTM) status including phosphorylation, nitration, and ubiquitination via traditional mass spectrometry analysis. PPG Core B will utilize the up-to-date technologies to provide a proteomic-genomic study platform for all three Projects and generate novel information on the genes targeted for investigation in this PPG. This PPG Core will also provide software and hardware infrastructure for (i) federated tissue management (e.g. chain of custody while transferring tissues from any lab to any Core) and (ii) secure data exchange between Cores and Research investigators. Core B will also resource investigators and Cores with TissueMetrix tissue management software that also provides chain of custody management of samples transferred between investigators and Cores and reconciliation between lists of samples send and received. In addition, large files generated by the cores will be transferred to investigators using a secure IPSwitch portal that also manages authentication and keeps logs of accessed files by legitimate users (e.g. genomics, transcriptomic, proteomics, imaging).
Each of the PPG projects focuses on a specific cytoskeletal target, but they all share similar regulation via genetic and post-translational modification. Core B will serve as a centralized infrastructure for providing expertise in the genomic and proteomic analysis of target DNA, RNA, and protein regulation.
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