National Heart, Lung, and Blood Institute

Proteomics Centerat

The University of Texas Medical Branch

atGalveston, 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

MarbanMarban - - JHUJHU

CostelloCostello - - Boston UBoston U

GreeneGreene - - MCWMCW

Kodadek Kodadek - - UT SouthwesternUT Southwestern

KuroskyKurosky- UT Galveston- UT Galveston

AebersoldAebersold - ISB- ISB

PollardPollard - - HMJFAMMHMJFAMM

WilliamsWilliams - Yale- Yale

NolanNolan- Stanford- Stanford

KnappKnapp -USC-USC

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 - ProgeneraGel 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 onimage

Key features and benefits – spot detection

• Automated spot detection– Removes subjectivity from 2DE

– Reproducible

– Robust

– Enabling technology

Progenera Features

• 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, MDProfessor of MedicineBiology Team Leaderarbrasie@utmb.edu

Biology Teams I-IIIBiology 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, MDViral Bronchiolitis Roberto Garofalo, MD

Airway epithelium: initiates the inflammatory response

Normal functions:Cellular barrier for gas exchangeMucociliary clearanceSecretes protective ELF

Stimulated:Mucus glycoprotein secretionProstaglandins/LeukotrienesCXC Chemokines (IL-8, GRO)CC chemokines

(RANTES, MCP-1, MIP-1

VirusVirus

Respiratory Syncytial Virus (RSV)

• Ubiquitous Negative-sense RNA virus for Ubiquitous Negative-sense RNA virus for which no vaccine is available.which no vaccine is available.

• Causative agent of Causative agent of – Epidemic wheezing in childrenEpidemic wheezing in children– Otitis MediaOtitis Media– Outbreaks in elderly and Outbreaks in elderly and

immunocompromisedimmunocompromised• Replicates in airway mucosa, producing Replicates in airway mucosa, producing

inflammation. inflammation.

Chemokine Expression patterns:

RSV induced inflammatory mediatorsRSV induced inflammatory mediators

IB Kinase IKK

IB

Rel A:NF-B1PhosphorylationDegradation

Rel A:NF-B1

nucleus

IB Kinase is Central Regulator of Inflammatory Responsecytokines

RSV

Membranecomplexes

Broad Goals: Cellular Inflammation• Identify components of the IKKIdentify components of the IKK

– Affinity purification/LC-MSAffinity purification/LC-MS

• Determine cytoplasmic, nuclear and membrane Determine cytoplasmic, nuclear and membrane proteome of airway epithelial cells. proteome of airway epithelial cells. – Optimize subcellular fractionation for reproducible Optimize subcellular fractionation for reproducible

2D gels/Mass fingerprinting2D gels/Mass fingerprinting– Estimate sample variationEstimate sample variation– Compare to microarray databaseCompare to microarray database

• Analyze cell cycle effects on proteomeAnalyze cell cycle effects on proteome• Determine effects of viral infection on Determine effects of viral infection on

subcellular organellar proteomessubcellular organellar proteomes

Thioaptamers as a Proteomics Tool

• Aptamers with thiophosphate backbone – Aptamers with thiophosphate backbone –

ThioaptamersThioaptamersTM TM

• In vitro and split synthesis combinatorial In vitro and split synthesis combinatorial librarieslibraries

• High-throughput screening and selectionHigh-throughput screening and selection

• Thioaptamer proteomics chips/beads (MS and Thioaptamer proteomics chips/beads (MS and optical)optical)

CK-14 XBY-2 XBY-6

NMR Structures of NF-B Aptamers

Summary: Thioaptamer Summary: Thioaptamer Advantages for ProteomicsAdvantages for Proteomics

• Very high affinity – nM Very high affinity – nM

• Very high specificity – NF-Very high specificity – NF-B single protein target binding B single protein target binding

• In vitroIn vitro combinatorial monothioate enzymatic selection combinatorial monothioate enzymatic selection

• Split synthesis combinatorial bead libraries – dithioate aptamersSplit synthesis combinatorial bead libraries – dithioate aptamers

• HTS via multicolor flow cytometry of thioaptamer bead librariesHTS via multicolor flow cytometry of thioaptamer bead libraries

• Greater stability towards nucleasesGreater stability towards nucleases

• Indefinite shelf-lifeIndefinite shelf-life

• Inexpensive to produce either synthetically or enzymaticallyInexpensive to produce either synthetically or enzymatically

• High reproducibility in quality control High reproducibility in quality control

• Rapid production of new thioaptamers – even proteome screen Rapid production of new thioaptamers – even proteome screen

• High success rate for selecting thioaptamers (7/7)High success rate for selecting thioaptamers (7/7)

• MALDI MS and optically detected thioaptamer proteomics chip? MALDI MS and optically detected thioaptamer proteomics chip?

Acknowledgement

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