# Presentation: 210010

## NHLBI Proteomics Initiative

**NHLBI Proteomics 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 Initiative

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

## NHLBI Proteomic Initiative

**Marban**
-  - *JHU*

**Costello**
-     - *Boston U*

**Greene**
-  - *MCW*

**Kodadek **
- - *UT Southwestern*

**Kurosky**
*	**- UT Galveston*

**Aebersold**
-  *- ISB*

**Pollard**
-  - *HMJFAMM*

**Williams**
-  *- Yale*

**Nolan**
*	**- Stanford*

**Knapp**
-     *-MUSC*

## Cardiovascular Proteomics Center

_**Mission**_** **
**The BUSM CPC undertakes innovative technology**
**development and highly integrated bioinformatics**
**approaches to characterize oxidative stress in cardiovascular**
**disease.**
**Director: Catherine Costello, Ph.D.**
**Co-PI: Richard Cohen, Ph.D.**
_**Key Personnel**_
- Takeshi Adachi 	 	Michael Kirber 		Flora Sam
- Emelia Benjamin		Martin  Larson 		Douglas Sawyer
- James Collins		Jane Leopold 		Martin Steinberg
- Wilson Colucci	 	Ronglih Liao  		Joseph Vita
- Harrison Farber	 	Joseph Loscalzo 		Kenneth Walsh
- Jane Freedman 		Mark McComb 		Zhipeng Weng
- Diane Handy		Peter O’Connor 		Stephen Yeung
- John Keaney 	         	V. Ramachadran
**www.bumc.bu.edu/cardiovascularproteomics**

## Cardiovascular Proteomics Center

- _Goals/Aims of Center_
- To develop new methodologies, hardware and software for exploration of oxidative stress
- To screen for alterations in protein abundances
- To investigate structural details of modified proteins
- To elucidate modifications due to oxidant stress
- To use bioinformatics techniques to identify network nodes that affect cellular response and represent potential therapeutic targets
**www.bumc.bu.edu/cardiovascularproteomics**

## Cardiovascular Proteomics Center      

- _Technology Development_
- High throughput MALDI Cryo-FTMS with data-dependent targeting of protein modifications
- Multistage capLC separations, new reagents
- Highly integrated bioinformatics approach
- _Biological Applications_
- Endothelial dysfunction
- Antioxidant deficiency states
- Proteomic markers in community, in diseases
**www.bumc.bu.edu/cardiovascularproteomics**

## Medical University of South CarolinaNHLBI Cardiovascular Proteomics Center

_**Mission**_** **
**To develop new technologies for proteomic analysis in the context of applying these technologies to studies of cardiac development, cardiac hypertrophy, and insulin resistance syndrome.**
**Director: Daniel R. Knapp, Ph.D.**
_**Key Personnel**_
- Jonas  Almeida, Ph.D. 	John M. Arthur, M.D., Ph.D.
- W. Timothy Garvey , M.D. 	Edward L. Krug Ph.D.
- Roger R. Markwald, Ph.D.  	Kevin L. Schey, Ph.D.
- Eberhard O.  Voit, Ph.D. 	Michael R. Zile, M.D.

## Medical University of South CarolinaNHLBI Cardiovascular Proteomics Center

_**Goals/Aims of Center**_
- Define proteomic changes associated with:
- • Diastolic heart failure
- (volume and pressure overload hypertrophy in rat model)
- •  Early cardiac development
- (epithelial  to mesenchymal transformation in mouse model)
- •  Insulin resistance syndrome
- (human skeletal muscle in metabolically defined subgroups)
- Develop improved technologies for proteomic analysis:
- • Improved 2D gel electrophoresis-based methods
- • Microfluidic devices for proteomic analysis
- • Mass spectrometry of intact proteins
- • Mathematical modeling of  proteomic systems

## Medical University of South CarolinaNHLBI Cardiovascular Proteomics Center

_**Technologies being applied:**_
**Subcellular fractionation**
**Two dimensional gel electrophoresis**
**Two dimensional liquid chromatography**
**Microfluidic devices**
**Electrospray ionization (ESI) mass spectrometry (ion trap, time of flight, quadrupole-time of flight)**
**Matrix-assisted laser desorption ionization (MALDI) mass spectrometry (time of flight, tandem time of flight)**
**Isotope coded affinity tags (ICAT)**
**Biochemical systems theory**
**Advanced numerical methods  (neural computing, genetic algorithms, stiff solvers)**
**XML-based data management**

## SouthwesternProteomic Center

_**Mission**_
- To develop technology for the massively parallel analysis of regulatory proteins (membrane receptors, transcription factors) and apply these tools to understanding the molecular basis of sleep.
**Director: Tom Kodadek, Ph.D.**
_**Key Personnel**_
- Stephen A. Johnston
- Skip Garner
- David Fancy
- Ross Chambers
- Masashi Yanagisawa

## SouthwesternProteomic Center

**Goals/Aims of Center**
*Medium-term goal*:
- Elucidate the molecular basis of orexin signaling
- Use various array tools to characterize important signal transduction and transcription factors involved in this process.
*Long-Term Goals*:
- Assay in relevant tissue
-  Arrangement of TFs.
-  Pattern of GPCR activation.
-  Levels and post-translational modification state of signal transduction Proteins.

## SouthwesternProteomic Center

_**Major Technology Goals**_
- Construct protein-detecting microarrays consisting of synthetic capture agents.  Quantify protein capture with a sandwich assay using labeled antibodies produced by genetic immunization.
- Construct arrays of functional GPCRs suitable for screeningextracts for neuroregulatory hormones involved in sleep homeostasis  as well as libraries for synthetic agonists and antagonists.
- Elaborate genome-wide chromatin immunoprecipitation (ChIP) and related assays as tools to identify changes in TF binding Patterns.

## Yale/NHLBI Proteomics Center

_**Mission**_
**To improve existing/develop new approaches to diagnose, more accurately classify, understand, and treat diseases related to vascular biology, hematopoiesis, and blood pressure regulation.**
- Director: Kenneth R. Williams, Ph.D.
- Co-Director: William C. Sessa, Ph.D.
- _http://info.med.yale.edu/nhlbi-proteomics/_

## Yale/NHLBI Proteomics Center

_**Key Personnel**_
_***MS, Protein Chemistry & Protein Profiling***_
**R. Breaker, C. Horvath, W. Konigsberg, K. Stone, W. McMurray & K. Williams**
_***Biostatistics, Bioinformatics & Database Design***_
**K. Cheung, M. Gerstein, P. Miller & H. Zhao**
_***Cell-Permeable, Synthetic Biotechnologies for Blocking Specific Protein-Protein Interactions***_
**F. Giordano, S. Ghosh, J. Pober, A. Schepartz, W. Sessa & D. Ward**
_***Hematopoiesis***_
**N. Berliner, F. Forget, K. Krause, A. Perkins & S. Weissman**
_***Hypertension***_
**R. Lifton**
_***Vascular Biology***_
**J. Bender & W. Sessa**
- _http://info.med.yale.edu/nhlbi-proteomics/_

## Yale/NHLBI Proteomics Center

**Goals/Aims of Center**
- Improve and develop new protein profiling biotechnologies to identify proteins that play key roles and/or serve as biomarkers for diseases such as atherosclerosis, hypertension, inflammation, blood diseases, and immunological rejection of transplanted tissues/organs.
- Design cell permeable, synthetic peptide-based reagents to block protein-protein interactions of key proteins involved in these disease processes in cells and tissues of interest.
- Utilize protein profiling to monitor the impact of blocking key protein-protein interactions in vivo.
-  _http://info.med.yale.edu/nhlbi-proteomics/_

## Yale/NHLBI Proteomics Center

**Technology Development**
- MALDI-MS based protein disease biomarker analysis of serum and other biological fluids.
- Quantitative ICAT/LC-MS/MS protein profiling.
- Differential (fluorescence) 2D gel electrophoresis protein profiling.
- Phosphoproteome analysis based on MS analysis of phosphopeptide-enriched fractions from digests of cell extracts.
- An Oracle based proteomics database is being developed to archive, retrieve, analyze, and cross- correlate protein profiling data with mRNA expression, functional, protein interaction, and other relevant data available in other databases.
- Development of ribozyme-based molecular biosensors for large scale analysis of _selected_ proteins and their post-translational modifications.
-  _http://info.med.yale.edu/nhlbi-proteomics/_

## Center for Medical Proteomics

_**Mission**_
**The Center for Medical Proteomics develops innovative technologies focused on discovery of disease biomarkers and therapeutic targets for Cystic Fibrosis.**
**Director: Harvey B. Pollard, M.D., Ph.D.**
- Project Leaders:
- Dr. Greg Mueller			Dr.Ofer Eidelman
- Dr. Meera Srivastava		Dr. David Jacobowitz
- Dr. Tom Darling 			Dr. Lee Metcalf
- Dr. William Guggino	 		Dr. Pamela Zeitlin
- htpp://www.usuhs.mil/APG

## Goals/Aims of Center

To identify all proteins in cultured CF lung epithelial cells which are altered by CF, using the technique of 3-D Proteomics. 
To identify all proteins in patient samples of CF lung epithelial cells which are affected by the disease using 3-D Proteomics.
To develop a CF proteomic chip using information from CF cells and tissues.

htpp://www.usuhs.mil/APG

- To identify all proteins in cultured CF lung epithelial cells which are altered by CF, using the technique of 3-D Proteomics.
- To identify all proteins in patient samples of CF lung epithelial cells which are affected by the disease using 3-D Proteomics.
- To develop a CF proteomic chip using information from CF cells and tissues.
- htpp://www.usuhs.mil/APG

**Center for Medical Proteomics**

## Center for Medical Proteomics

**Technology Development**
- 3-D Proteomics
- Laser Capture Microdissection (LCM)
- Free Flow Electrophoresis (FFE) for protein separation
- High Throughput Screen (HTS) for disease biomarkers
- Bioinformatic tool development (“PROT-SAVER”)
**Biological Application**
- Identification of biomarkers for CF diagnostics.
- Identification of surrogate endpoints for CF drug development
- Identification of drug targets for CF therapeutics
- htpp://www.usuhs.mil/APG

## Development of Novel Mass Spectrometry Tools for Individual Cell Proteome Analysis (Medical College of Wisconsin in Milwaukee)

**Development of Novel Mass Spectrometry Tools for Individual Cell Proteome Analysis**** ****(****Medical College of Wisconsin in Milwaukee)**

_**Mission**_
**To develop novel mass spectrometric methodologies and technology for the quantitative analysis of the entire proteome from samples as small as individual cells.  A comprehensive approach to sample generation, technology development, and data analysis will generate not only new instrumentation but also important physiological data that will be rapidly and freely disseminated.**
**Director: Andrew S. Greene**
- _Key Personnel_
- Lloyd M. Smith	Peter J. Tonellato    Michael Olivier
- J. Michael Ramsey       Michael S. Westphall
- http://proteomics.mcw.edu

## Goals/Aims of Center

Proteomics and Technology Development:  Development of novel mass spectrometric methodologies and technology for the quantitative analysis of the entire proteome of a single cell. 

Animal Models and Experimental Systems:  The Animal Models and Experimental Systems Component, is charged with the development, maintenance and production of the animal and cell culture models of angiogenesis to be studied.  

Protein analysis and Technology Implementation:  To implement the technological advances in analytical mass spectrometry (component 1) by merging them with existing analytical capabilities to identify and quantify the proteome of individual cells or tissue samples and cell lines during angiogenesis. 

Bioinformatics: To apply state-of-the-art information science techniques to applications in proteomics. 

Administration:  To integrate and facilitate the activities of the scientific components. 
http://proteomics.mcw.edu

**Proteomics and Technology Development**:  Development of novel mass spectrometric methodologies and technology for the quantitative analysis of the entire proteome of a single cell.
**Animal Models and Experimental Systems**:  The Animal Models and Experimental Systems Component, is charged with the development, maintenance and production of the animal and cell culture models of angiogenesis to be studied.
**Protein analysis and Technology Implementation**:  To implement the technological advances in analytical mass spectrometry (component 1) by merging them with existing analytical capabilities to identify and quantify the proteome of individual cells or tissue samples and cell lines during angiogenesis**. **
**Bioinformatics**: To apply state-of-the-art information science techniques to applications in proteomics.
**Administration**:  To integrate and facilitate the activities of the scientific components.
- _http://pro_teomics.mcw.edu

**Development of Novel Mass Spectrometry Tools for Individual Cell Proteome Analysis**** ****(****Medical College of Wisconsin in Milwaukee)**

## Technology Development
To develop new instrumental approaches to the mass spectrometric analysis of complex mixtures of proteins and multi-subunit protein complexes, with the goal of dramatically increasing the sensitivity, resolution, and mass range of the analysis process. In tandem with this effort, we will develop approaches for the processing of individual cells into a form suitable for mass spectrometric analysis of their components. 

Biological Application
To identify and characterize proteins involved in the angiogenic phenotype in microvascular endothelial cells derived from a set of consomic rats.

http://proteomics.mcw.edu

- To develop new instrumental approaches to the mass spectrometric analysis of complex mixtures of proteins and multi-subunit protein complexes, with the goal of dramatically increasing the sensitivity, resolution, and mass range of the analysis process. In tandem with this effort, we will develop approaches for the processing of individual cells into a form suitable for mass spectrometric analysis of their components.
**Biological Application**
- To identify and characterize proteins involved in the angiogenic phenotype in microvascular endothelial cells derived from a set of consomic rats.
- http://proteomics.mcw.edu

**Development of Novel Mass Spectrometry Tools for Individual Cell Proteome Analysis**** ****(****Medical College of Wisconsin in Milwaukee)**

## Seattle Proteome Center

_**Mission**_
**To develop an array of new, systematic assays to comprehensively study the dynamics of cellular proteomes in health and disease.**
**Director: Ruedi Aebersold,  Ph.D.**
_**Key Personnel**_
- Elaine Raines
- David Goodlett
- Alan Aderem
- http://www.proteomecenter.org

## Seattle Proteome Center

_**Goals/Aims of Center**_
- To develop an integrated technology platform to quantitatively and systematically determine different properties of proteins;
- To apply the newly developed technologies to a broad range of biological research projects and to widely disseminate them;
- To apply quantitative proteomics technologies to advance research into heart, blood and lung in health and disease;
- To promote continual interplay between biology and technology resulting in ongoing testing and refinement of new proteomic technologies
- http://www.proteomecenter.org

## Seattle Proteome Center

- Technology Development
- The approach pursued by the SPC is mass spectrometry-based quantitative proteome analysis.  Technology development is structured into ten technology modules that collectively cover the whole analytical process from sample preparation to data analysis and management.  These modules involve research in the fields of chemistry, biology, protein chemistry, the separation sciences, mass spectrometry and computer science.
- http://www.proteomecenter.org

## Seattle Proteome Center

- Biological Applications
- The biological focus of the SPC is the macrophage.  In the first stage, specific properties of macrophage biology (e.g., signaling) will be explored.
- In the second phase, macrophage biology will be systematically studied using a combination of genomic and proteomic approaches.
- http://www.proteomecenter.org

## Proteomic Analysis of Blood Components in Autoimmune Disease (Stanford University)

**Proteomic Analysis of Blood Components ****in Autoimmune Disease**** ****(Stanford University)**

_**Mission**_
- To expand an understanding of the auto-immune diseases Rheumatoid Arthritis, System Lupus Erythematosus, and related disorders, using novel proteomics technologies that allow cutting-edge cell-based and blood-based analysis.
***Director:  Garry P. Nolan, Ph.D.***
_**Key Personnel**_
**	****Paul J Utz, M.D., Ph.D.****	****Gilbert Chu, M.D., Ph.D.**
**	****Larry Steinman, M.D.****	****William Robinson, M.D., Ph.D.**
**	****Robert Tibshirani, Ph.D.****	****Juan Santiago, Ph.D.**

## Proteomic Analysis of Blood Components in Autoimmune Disease (Stanford University)

_**Goals/Aims of Center**_
- Apply leading edge flow cytometry single-cell based technologies for multiparameter analysis of signaling systems based on phosphoprotein status.
- Multiplexed quantitation and characterization of secreted molecules via advanced capillary electrophoresis systems.
- Develop antigen microarray technologies for auto-antibody profiling in auto-immune diseases.
- Development of relational software and statistical analysis regimens that will allow the comparison and correlation of different datasets generated by diverse proteomic and genomic technologies.
- Use the above approaches to develop proteomic signatures for hypothesis generation and disease prevention.

## Proteomic Analysis of Blood Components in Autoimmune Disease (Stanford University)

_***Technology Development***_
- Flow Cytometry phospho-protein analysis in complex populations of immune cells to study intracellular signaling
- Highly sensitive microcapillary based secreted protein detection
- Array based antigen profiling of antibodies during auto-immune progression.
- Cross-platform megaset statistical analysis regimes to merge correlations across diverse and large proteomics datasets
_***Biological Applications:***_
- Multiparameter Immune system signaling analysis
- Sensitive immune-profiling for secreted bio-agents
- Proteomic signatures for hypotheses and biological mechanism generation.

## NHLBI Proteomics Center at Johns Hopkins

_**Mission Statement**_
- To obtain “disease fingerprints” of numerous ischemic/hypoxic disorders, which may then provide a unified picture of their pathogenesis.
**Director: Eduardo Marb****án****, M.D., Ph.D.**
- _Key Personnel_
- 	G. Semenza	J. Van Eyk
- 	J. Garcia	G. Hart
- 	C. Dang	R. Cotter
- 	A. Murphy	R. Winslow
- 	R. Cole

**NHLBI Proteomics Center at ****Johns Hopkins**

## Technology Development/Biological Application

**NHLBI Proteomics Center at ****Johns Hopkins**

- Novel biological models of ischemia/ hypoxia
- Improved sample preparation
- Modifications of ICAT
- New mass spec methodologies
- Bioinformatics/ databasing

- Insights into mechanism and biomarkers
- Fuller representation of proteome
- Better identification  of PTMs
- Improved protein identification
- Patterns: clusters, time courses, disease changes

## University of Texas Medical Branch NHLBI Proteomic Center

- Mission Statement
-     This center will apply a comprehensive and innovative proteomic strategy using novel technologies that will be generally applicable to many NHLBI research interests. Specifically, the UTMB Center will employ proteomic-based technologies to investigate protein expression associated with airway inflammatory processes.
- 		Alexander Kurosky, Ph.D. (Director)
-      Allan Brasier, M.D. (Assoc. Director)        Roberto Garofalo, M.D.
-      David Gorenstein, Ph.D.                         James Leary, Ph.D.
-      Bruce Luxon, Ph.D.                                Sanjiv Sur, M.D.
-  http://www.bioinfo.utmb.edu/proteomics/NHLBI

## University of Texas Medical Branch NHLBI Proteomic Center

- Major Center Goals
- Develop sensitive high-resolution protein separation technologies to construct protein databases of changes in cellular and subcellular protein expression in the major cell types of the inflamed airways.
- Develop innovative thioaptamer-based array and bead technologies to identify and quantify proteins and protein complexes associated with critical signaling and immune response pathways relating to airway inflammation.
- Apply specialized high-throughput flow cytometry/cell sorting and laser-capture micro dissection for subsequent proteome analysis of defined cell types in specific stages of the cell cycle.
- Conduct systems modeling to correlate changes in protein expression with available genomic data on changes in gene expression due to inflammation after respiratory syncytial virus infection or asthma.
-   http://www.bioinfo.utmb.edu/proteomics/NHLBI

## University of Texas Medical Branch NHLBI Proteomic Center

- Technology Development
- High-performance 2D gel analysis combined with novel image analysis methodology.
- Thioaptamer array technologies for diagnostic and therapeutic benefit.
- Innovative high speed cell sorting/selection.
- Inflammatory pathways modeling.
- Biological Application
- Cellular, animal, and human models of viral- and allergen-induced asthma
-    http://www.bioinfo.utmb.edu/proteomics/NHLBI

## Technology Development

- Protein Abundance
- Modification detection
- 2D Separation Technology
- Microfluidic Devices
- Mass Spectrometry of intact proteins
- Modeling of Proteomic Systems
- Protein Detecting Microarrays
- Synthetic Capture Agents

## Technology Development

- Protein-Protein Interactions
- Protein Profiling
- 3-D Proteomics
- Free Flow Electrophoresis
- Mass Spectrometry Analysis of Complex Mixtures
- Flow Cytometry
- Microcapillary based Secreted Protein Detection
- Array Based Antigen Profiling

## Biological Applications

- Oxidative Stress in Cardiovascular Disease
- Cardiovascular Development
- Cardiac Hypertrophy
- Insulin Resistance
- Vascular Biology
- Hematopoiesis
- Blood Pressure

## Biological Application

- Sleep
- Cystic Fibrosis
- Macrophage Biology
- Auto-immune Diseases
- Immune Signaling
- Ischemic/Hypoxic Disorders
- Inflammation of the Airways

## Goals

- Systematic assays to comprehensively study the dynamics of cells in health and disease
- Quantitative analysis of the entire proteome of individual cells
- Develop technology for the massively parallel analysis of regulatory proteins
- Develop new approaches to diagnose, classify, understand and treat disease
- Innovative technologies focused on discovery of disease biomarkers and therapeutic targets
- Obtain disease fingerprints