1. Introduction to Proteomics

2. What is Proteomics?
Proteomics - A newly emerging field of life science research that uses High Throughput (HT) technologies to display, identify and/or characterize all the proteins in a given cell, tissue or organism (I.e. the proteome).

3. 3 Kinds of Proteomics Expressional Proteomics Functional Proteomics
Electrophoresis, Protein Chips, DNA Chips, SAGE
Mass Spectrometry, Microsequencing
Functional Proteomics
HT Functional Assays, Ligand Chips
Yeast 2-hybrid, Deletion Analysis, Motif Analysis
Structural Proteomics
High throughput X-ray Crystallography/Modelling
High throughput NMR Spectroscopy/Modelling

4. Expressional Proteomics
2-D Gel QTOF Mass Spectrometry

5. Expressional Proteomics
Prostate tumor Normal

6. Expressional Proteomics

7. Why Expressional Proteomics?
Concerned with the display, measurement and analysis of global changes in protein expression
Monitors global changes arising from application of drugs, pathogens or toxins
Monitors changes arising from developmental, environmental or disease perturbations
Applications in medical diagnostics and therapeutic drug monitoring

8. Examples
Jungblut PR et al., “Proteomics in Human Disease: Cancer, Heart and Infectious Disease” Electrophoresis 20: (1999)
Zhukov TA et al., “Discovery of distinct protein profiles specific for lung tumors and pre-malignant lung lesions by SELDI” Lung Cancer 40: (2003)
Ghaemmaghami S, et al., “Global analysis of protein expression in yeast” Nature 425: (2003).

9. Functional Proteomics

10. Functional Proteomics (in vitro)
Multi-well plate readers
Full automation/robotics
Fluorescent and/or chemi-luminescent detection
Small volumes (mL)
Up to 1536 wells/plate
Up to 200,000 tests/day
Mbytes of data/day

11. Functional Proteomics

12. Functional Proteomics
In silico methods (bioinformatics)
Genome-wide Protein Tagging
Genome-wide Gene Deletion or Knockouts
Random Tagged Mutagenisis or Transposon Insertion
Yeast two-hybrid Methods
Protein (Ligand) Chips

13. Why Functional Proteomics?
Concerned with the identification and classification of protein functions, activities locations and interactions at a global level
To compare organisms at a global level so as to extract phylogenetic information
To understand the network of interactions that take place in a cell at a molecular level
To predict the phenotypic response of a cell or organism to perturbations or mutations

14. Examples
Uetz P et al., “A Comprehensive Analysis of Protein-Protein Interactions in Saccharomyces cerevisiae” Nature 403: (2000)
First example of whole proteome analysis
957 putative interactions
1004 of 6100 predicted proteins involved

15. Examples
Huh, K et al., “Global analysis of protein localization in budding yeast” Nature, 425: (2003)
Used a collection of yeast strains expressing full-length, chromosomally tagged green fluorescent protein (GFP) fusion proteins
Localized 75% of the yeast proteome, into 22 distinct subcellular localization categories
Provided localization information for 70% of previously unlocalized proteins

16. Examples
Edwards JS & Palsson BO “Systems properties of the H. influenzae Rd metabolic genotype” J. Biol. Chem. 274: (1999)
First example of metabolic/phenotypic prediction using proteome-wide information
Converting sequence data to differential equations so as to predict biology/behavior

17. Structural Proteomics
High Throughput protein structure determination via X-ray crystallography, NMR spectroscopy or comparative molecular modeling

18. Structural Proteomics: The Goal

19. Structural Proteomics: The Motivation
200000
180000
160000
140000
120000
Sequences
100000
Structures
80000
60000
40000
20000

20. The Protein Fold Universe
500?
2000?
10000?
How
Big Is
It??? 8 ?

21. Protein Structure Initiative
Organize all known protein sequences into sequence families
Select family representatives as targets
Solve the 3D structures of these targets by X-ray or NMR
Build models for the remaining proteins via comparative (homology) modeling

22. Protein Structure Initiative
Organize and recruit interested structural biologists and structure biology centres from around the world
Coordinate target selection
Develop new kinds of high throughput techniques
Solve, solve, solve, solve….

23. Why Structural Proteomics?
Structure Function
Structure Mechanism
Structure-based Drug Design
Solving the Protein Folding Problem
Keeps Structural Biologists Employed

24. Structural Proteomics - Status
20 registered centres (~30 organisms)
82700 targets have been selected
52705 targets have been cloned
29855 targets have been expressed
12311 targets are soluble
1493 X-ray structures determined
502 NMR structures determined
1743 Structures deposited in PDB

25. Structural Proteomics - Status
543 structures deposited by Riken
265 structures deposited by Mid-West
187 structures deposited by North-East
179 structures deposited by New York
178 structures deposited by JCSG (UCSD)
52 structures deposited by Berkeley
31 structures deposited by Montreal/Kingston

26. Bioinformatics & Proteomics
Agriculture
Medicine
Bioinformatics
Proteomics
Genomics

27. Bioinformatics & Functional Proteomics
How to classify proteins into functional classes?
How to compare one proteome with another?
How to include functional/activity/pathway information in databases?
How to extract functional motifs from sequence data?
How to predict phenotype from proteotype?

28. Bioinformatics & Expressional Proteomics
How to correlate changes in protein expression with disease?
How to distinguish important from unimportant changes in expression?
How to compare, archive, retrieve gel data?
How to rapidly, accurately identify proteins from MS and 2D gel data?
How to include expression info in databases?

29. Bioinformatics & Structural Proteomics
David Wishart
June 2005
Bioinformatics & Structural Proteomics
How to predict 3D structure from 1D sequence?
How to determine function from structure?
How to classify proteins on basis of structure?
How to recognize 3D motifs and patterns?
How to use bioinformatics databases to help in 3D structure determination?
How to predict which proteins will express well or produce stable, folded molecules?
(c) 2005 CGDN

30. Homework Download RASMOL Download PDB file from Protein Data Bank
Provide functional protein information/characteristics from the PDB file as opened using RASMOL
Characteristics
Protein name
Sequence
Number of:
Chains
Bonds
Amino acids
Alpha helices
Beta strands