Proteomics – Analysis of Selected Emerging Technologies

Table of Contents

 

1.      Introduction – the Current State of Proteomic Technology

 

1.1  A Larger Task Ahead

 

1.2   Industrialization of Mature Technologies

 

Incremental Improvements

 

Industrialization

 

1.3  Seeking Alternatives to Traditional Proteomics

 

1.4  The Future is Looking Very Small

 

1.5 Complementary Technologies Enabling Chips

 

1.6 Beating a Path to New Drugs

 

1.7 Post-Proteomics

 

1.8  Diagnostics is the Earliest Beneficiary of Proteomics

 

1.9 A Compelling Need for Another Industry-Wide Initiative

 

 

2.   Impact of Automation on Proteomics

Presents several key components of automation in proteomics technology, and the improvements required to obtain not simply more information, but high quality information. This is critical for a complex “system” such as the proteome, where inexact results could lead researchers down many wrong paths in discovery.

 

2.1 Proteomics Relies on Automation

 

2.2 A Comparison of Complex Systems

 

2.3 Current Limitations to Proteomic Analysis and Automation

 

2.4 Tandem Affinity Purification

 

2.5 Automating TAP Purification and Analysis

 

2.6 Case Example in the Identification of Protein-Protein Interactions

 

2.7 Capturing Transient Interactions

 

2.8 What Lies Ahead in Proteomic Automation

 

 

3.      Convergent Solutions to Binding at a Protein-Protein Interface

The rapidly growing number of protein structures is permitting an examination to reveal the consensus characteristics of protein-protein interactions. These characteristics are described here.  Because so many disease pathways involve interactions between proteins, the implications of deciphering the rules of interaction on the development of peptide and small molecule therapeutics can be significant.

 

3.1 A Business Model at the Interface of Proteomics and Drug Discovery

 

3.2 Prelude to Drug Discovery – Deciphering the Rules of Protein-Protein Interaction

 

3.3 Seeking Small Molecule Binding at the Protein Interface

 

 

4.      Finding Inhibitors of Protein-Protein Interactions

Functional proteomics is yielding large databases of interacting proteins and extensive pathway maps of these interactions are being deciphered by novel high throughput technologies. However, traditional methods of screening have not been very successful in identifying protein-protein interaction inhibitors. Described in this chapter are technologies used to rapidly screen for compounds that inhibit protein-protein interactions, including allosteric inhibitors.

 

4.1 An Industry Need to Decipher Protein Function

 

4.2 A Platform Technology for Deciphering Protein Function

 

4.3 Case Study in the Identification of a Receptor Inhibitor

 

4.4 Validating Targets with Functional Interactive Screening Technology

 

4.5 A System for Detecting Heterodimer Protein-Protein Interactions

 

4.6 Merging Biological Assays and Advanced Chemistry to Find Drug Prospects

 

 

 

5.      Protein Expression Profiling on Microarrays by Rolling Circle Amplification

Rolling circle amplification (RCA) is emerging as the signal amplification method of choice for DNA and RNA microarrays. A modification of RCA enables protein expression profiling on microarrays.

 

5.1 What is Rolling Circle Amplification?

 

5.2 Signal Amplification for Proteomics

 

5.4 The Benefits of Immuno-RCA to Proteomics and Diagnostics

 

5.5 Protein Chips

 

5.6 Application – Allergen Specific IgE Chip

 

5.7 Impact on Diagnostics

 

5.8 Application – Protein Expression Profiling

 

5.9 Comparison of Protein Chip with Conventional Proteomics

 

5.10 In Situ Detection An Application of Immuno-RCA Protein Detection

 

5.11 A Business Model Based on a Broadly Applicable Technology

 

 

6.      Protein Chips and Phage Display

Discusses the use of phage display technology to bind the proteome onto a chip. By coupling phage display’s capability of generating diverse libraries of human antibodies in vitro and protein chip microarrays, the two technologies can potentially fill a large gap in current approaches to proteomics. Applications enable recognition of protein modifications and differential expression of hundreds of proteins at once.

 

6.1 Phage Display Matching the Demand for Disease Target Analysis

 

6.2 Exploiting the Combination of Phage Display and Microarray Technologies

 

6.3 Description of the Technology

 

6.4 Source of Targets for Phage Display

 

6.5 Applications of Phage Selected Antibodies

 

6.6 Phage Display and Microarrays -- Monitoring Functionally Relevant Proteins

 

6.7 Application to Identification of Apoptosis Targets

 

6.8 Dyax Business Strategy

 

 

7.      Application of Novel Protein Chip Technology to Human Disease

Protein chips are an emerging technology useful for the discovery and measurement of protein biomarkers for therapeutic and diagnostic applications. The hope is that they will replicate some of the remarkable capabilities that DNA microarrays brought to the field of genomics. Examples of SELDI  protein chip technology are presented  along with their use in the discovery, identification and characterization of protein biomarkers for prostate cancer and other diseases.

 

7.1 Fishing for Therapeutic and Diagnostic Targets

 

7.2 How Protein Chip Arrays Work

 

7.3 Application – Protein Mining

 

7.4 Application – Disease Target and Marker Discovery

 

7.5 Application – Protein Identification and Purification

 

7.6 Cluster Analysis to Identify Complex Patterns in Multiple Mass Spectra

 

7.8 Application – Assay Development

 

7.9 Conclusion

 

8.  Identification of Kinase Targets from Proteomic Libraries using ProFusion Technology

A discussion on the method of covalently linking proteins to their mRNA, creating a link between protein and genotype. The protein moiety can be selected for under the most robust conditions and its genetic material amplified by PCR.. Profusion libraries can be constructed from the mRNA of any organism or tissue of interest, and screened for novel protein-protein, enzyme-substrate and protein-drug interactions.

 

8.1 mRNA/Protein Fusion

 

8.2 Profusion in Practice

 

8.3 Application – A Fibronectin Affinity Reagent Library

 

8.4 The Profusion Method Applied to Proteomics

 

8.5 Application – Identification of Novel Phosphorylation Targets and Inhibitors

 

8.6 From Phosphorylation Target to Phosphorylation Inhibitor

 

8.7 Application -- Protein-Protein Interactions in the Apoptotic Pathway

 

8.8 Application – Drug-Target Interaction

 

8.9 Blazing New Pathways

 

 

9.      Determination of Protein Functionality Using Live Cell Assays

Obtain functional information on a wide variety of cellular effects, including apoptosis, proliferation, differentiation, migration and protein secretion. Enables a better understanding of protein functionality in normal and diseased states.

 

9.1 Live Cell Assay Platform Technology

 

9.2 Examples of Monitoring Biological Endpoints in Live Cells

 

Motility

 

Proliferation

 

Apoptosis

 

Differentiation

 

Morphology

 

9.3 Quantifying Details of Cellular Behavior in Response to Proteins or Drugs

 

 

10. Proteomics Panel Discussion

 

Appendix. Corporate Technology Profiles and Collaborative Activity in Proteomics