Transcript Telehealth: Changing the Face of Health Care

National Heart, Lung, and
Blood Institute
Proteomics Center
at
The University of Texas Medical
Branch
at
Galveston, Texas
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.
NHLBI Proteomic
Initiative
• September 30, 2002
• Broad Agency Announcement
Contracts
• $157 million over 7 years
• 10 Proteomic Centers
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
Organizational Chart
UTMB NHLBI Proteomics Center
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
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
What is Progenesis?
It transforms the scope of this protein separation
technique, both in terms of throughput, reliability and
sensitivity
Click on
image
Key features and benefits – spot detection
• Automated spot detection
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Removes subjectivity from 2DE
Reproducible
Robust
Enabling technology
Progenera Features
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User specific front end
Sample tracking tool
2D analysis
Database administration + data security tool
Query and Data mining tools
Mass Spec. database query tool
Report tool
Statistical tools
• Comparing 2 populations:
– F-test (variance ratio test)
– t-Tests (one- and two-sided)
– Kolmogorov-Smirnov
• Comparing n populations:
– Kruskall-Wallis
• Comparing n treatments against a control
– Dunnett's
Graves and Haystead (2002)
Microbiol & Molec. Biol.
Rev. 66, 39-63
Gavin et al. (2002)
Nature 415, 141-147
Protein/Protein Interactions
Pull down experiments
- immunoprecipates
- thioaptamer beads
Tandem LC
2D-SDS-PAGE
Localization and co-localization with confocal
microscopy
Biology Teams:
Rationale and Approaches
Allan Brasier, MD
Professor of Medicine
Biology Team Leader
[email protected]
Biology Teams I-III
Team I:
Cellular Inflammation
Investigators:
Allan Brasier, MD
Antonella Casola, MD
Team II:
Mouse Inflammation
Roberto Garofalo, MD
Team III:
Human Inflammation
Viral Bronchiolitis
Sanjiv Sur, MD
Roberto Garofalo, MD
Airway epithelium:
initiates the inflammatory response
Normal functions:
Cellular barrier for gas exchange
Mucociliary clearance
Secretes protective ELF
Virus
Stimulated:
Mucus glycoprotein secretion
Prostaglandins/Leukotrienes
CXC Chemokines (IL-8, GROa)
CC chemokines
(RANTES, MCP-1, MIP-1a)
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.
RSV induced inflammatory mediators
Chemokine Expression patterns:
IkB Kinase is Central Regulator
cytokines of Inflammatory Response
RSV
IkB Kinase
IKK a,b
Membrane
complexes
IkBa
Rel A:NF-kB1
Phosphorylation
Degradation
Rel A:NF-kB1
nucleus
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
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)
NMR Structures of NF-kB Aptamers
CK-14
XBY-2
XBY-6
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?
Acknowledgement
• NCRR support in 2001 for a mass
spectrometer seeded UTMB’s
proteomic initiative