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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, Texas


Nhlbi proteomic initiative
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 initiative1
NHLBI Proteomic Initiative

  • September 30, 2002

  • Broad Agency Announcement Contracts

  • $157 million over 7 years

  • 10 Proteomic Centers


Nhlbi proteomic initiative2
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


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
Biology Teams:Rationale and Approaches

Allan Brasier, MD

Professor of Medicine

Biology Team Leader

[email protected]


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


Airway epithelium initiates the inflammatory response
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)


Respiratory syncytial virus rsv
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.


Chemokine Expression patterns:

RSV induced inflammatory mediators


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


Broad goals cellular inflammation
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
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
Acknowledgement

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


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