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National Heart, Lung, and Blood Institute Proteomics Center at The University of Texas Medical Branch at Galveston, Texa

National Heart, Lung, and Blood Institute Proteomics Center at The University of Texas Medical Branch at Galveston, Texas. NHLBI Proteomic Initiative.

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National Heart, Lung, and Blood Institute Proteomics Center at The University of Texas Medical Branch at Galveston, Texa

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  1. National Heart, Lung, and Blood Institute Proteomics Center at The University of Texas Medical Branch at Galveston, Texas

  2. NHLBI Proteomic Initiative The purpose of the NHLBI Proteomic Initiative is to establish local, highly interactive, multi-disciplinary Centers to enhance and develop innovative proteomic technologies and apply them to relevant biological questions that will advance our knowledge of heart, lung, blood, and sleep health and disease. This Initiative is intended to complement and enhance the NHLBI’s ongoing research programs, which include a substantial investment in clinical research, genomic research, basic biology, technologies, and training and education programs.

  3. NHLBI Proteomic Initiative • September 30, 2002 • Broad Agency Announcement Contracts • $157 million over 7 years • 10 Proteomic Centers

  4. NHLBI Proteomic Initiative Aebersold - ISB Greene - MCW Costello - Boston U Williams - Yale Marban - JHU Nolan - Stanford Pollard - HMJFAMM Kodadek - UT Southwestern Knapp -USC Kurosky - UT Galveston

  5. Organizational Chart UTMB NHLBI Proteomics Center

  6. Protein Identification • Applied Biosystems Voyager DE STR • MALDI-TOF • Micromass QTOF2 • Nanoflow LC ESI/MS/MS • Ciphergen • Surface enhanced laser desorption/ionization (SELDI) • Biology System III • PC1 1000 QTOF2 MALDI interface

  7. Gel Related Technologies • 1 & 2-D SDS PAGE • 1st dimension – Pharmacia ETTAN IPGphor • 2nd dimension – Biorad multiple gel systems (Protean Plus & Criterion) • Gel Imaging • Perkin Elmer ProXPRESS • Gel Analysis • Nonlinear Dynamics - Progenesis • - Progenera • Gel Robotics • Genomic Solutions - ProPic • - ProPrep

  8. Graves and Haystead (2002) Microbiol & Molec. Biol. Rev. 66, 39-63

  9. Gavin et al. (2002) Nature 415, 141-147

  10. Protein/Protein Interactions • Pull down experiments • - immunoprecipates • - thioaptamer beads • Tandem LC • 2D-SDS-PAGE • Localization and co-localization with confocal microscopy

  11. Biology Teams:Rationale and Approaches Allan Brasier, MD Professor of Medicine Biology Team Leader arbrasie@utmb.edu

  12. Biology Teams I-III Team I: Investigators: Cellular Inflammation Allan Brasier, MD Antonella Casola, MD Team II: Mouse Inflammation Roberto Garofalo, MD Team III: Human Inflammation Sanjiv Sur, MD Viral Bronchiolitis Roberto Garofalo, MD

  13. Airway epithelium: initiates the inflammatory response Virus • Normal functions: • Cellular barrier for gas exchange • Mucociliary clearance • Secretes protective ELF • Stimulated: • Mucus glycoprotein secretion • Prostaglandins/Leukotrienes • CXC Chemokines (IL-8, GROa) • CC chemokines • (RANTES, MCP-1, MIP-1a)

  14. Respiratory Syncytial Virus (RSV) • Ubiquitous Negative-sense RNA virus for which no vaccine is available. • Causative agent of • Epidemic wheezing in children • Otitis Media • Outbreaks in elderly and immunocompromised • Replicates in airway mucosa, producing inflammation.

  15. Chemokine Expression patterns: RSV induced inflammatory mediators

  16. IkB Kinase is Central Regulator of Inflammatory Response cytokines RSV IkB Kinase IKK a,b IkBa Membrane complexes Rel A:NF-kB1 Rel A:NF-kB1 Phosphorylation Degradation nucleus

  17. Broad Goals: Cellular Inflammation • Identify components of the IKK • Affinity purification/LC-MS • Determine cytoplasmic, nuclear and membrane proteome of airway epithelial cells. • Optimize subcellular fractionation for reproducible 2D gels/Mass fingerprinting • Estimate sample variation • Compare to microarray database • Analyze cell cycle effects on proteome • Determine effects of viral infection on subcellular organellar proteomes

  18. Thioaptamers as a Proteomics Tool • Aptamers with thiophosphate backbone – ThioaptamersTM • In vitro and split synthesis combinatorial libraries • High-throughput screening and selection • Thioaptamer proteomics chips/beads (MS and optical)

  19. NMR Structures of NF-kB Aptamers CK-14 XBY-2 XBY-6

  20. Summary: Thioaptamer Advantages for Proteomics • Very high affinity – nM • Very high specificity – NF-kB single protein target binding • In vitro combinatorial monothioate enzymatic selection • Split synthesis combinatorial bead libraries – dithioate aptamers • HTS via multicolor flow cytometry of thioaptamer bead libraries • Greater stability towards nucleases • Indefinite shelf-life • Inexpensive to produce either synthetically or enzymatically • High reproducibility in quality control • Rapid production of new thioaptamers – even proteome screen • High success rate for selecting thioaptamers (7/7) • MALDI MS and optically detected thioaptamer proteomics chip?

  21. Acknowledgement • NCRR support in 2001 for a mass spectrometer seeded UTMB’s proteomic initiative

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