Telehealth: Changing the Face of Health Care

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National Heart, Lung, and Blood
Institute Proteomics Center at The University of
Texas Medical Branch at Galveston, Texas
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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 NHLBIs
  ongoing research programs, which include a
  substantial investment in clinical research,
  genomic research, basic biology, technologies,
  and training and education programs.

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NHLBI Proteomic Initiative

• September 30, 2002
• Broad Agency Announcement Contracts
• 157 million over 7 years
• 10 Proteomic Centers

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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
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Organizational Chart UTMB NHLBI Proteomics Center
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• 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

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• 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

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Graves and Haystead (2002) Microbiol Molec.
Biol. Rev. 66, 39-63
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Gavin et al. (2002) Nature 415, 141-147
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• Protein/Protein
  Interactions
• Pull down experiments
• - immunoprecipates
• - thioaptamer beads
• Tandem LC
• 2D-SDS-PAGE
• Localization and co-localization with confocal
  microscopy

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Biology TeamsRationale and Approaches
Allan Brasier, MD Professor of Medicine Biology
Team Leader arbrasie_at_utmb.edu
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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
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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)

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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.

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Chemokine Expression patterns
RSV induced inflammatory mediators
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IkB Kinase is Central Regulator of Inflammatory
Response
cytokines
RSV
IkB Kinase IKK a,b
IkBa
Membrane complexes
Rel ANF-kB1
Rel ANF-kB1
Phosphorylation Degradation
nucleus
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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

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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)

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

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Acknowledgement

• NCRR support in 2001 for a mass spectrometer
  seeded UTMBs proteomic initiative