Proteomics

Micro 343 David Wishart Rm. Ath 3-41 david.wishart@ualberta.ca

Objectives
To gain awareness of what Proteomics is and what it isnt To become familiar with the different types if Proteomics To gain some basic understanding of the new tools being used in proteomics and to appreciate their strengths and weaknesses

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).

Proteomics
Proteomics employs an incredibly diverse range of technologies including:
molecular biology chromatography electrophoresis mass spectrometry X-ray crystallography NMR spectroscopy robotics computational biology

Proteomics Tools*
Molecular Biology Tools Separation & Display Tools Protein Identification Tools Protein Structure Tools

Molecular Biology Tools*

Northern/Southern Blotting Differential Display RNAi (small RNA interference) Serial Analysis of Gene Expression (SAGE) DNA Microarrays or Gene Chips Yeast two-hybrid analysis Immuno-precipitation/pull-down

DNA Microarrays*
Principle is to analyze gene (mRNA) or protein expression through large scale non-radioactive Northern (RNA) or Southern (DNA) hybridization analysis Brighter the spot, the more DNA Microarrays are like Velcro chips made of DNA fragments attached to a substrate Requires robotic arraying device and fluorescence microarray reader

Gene Chip Tools

DNA Microarrays

DNA Microarray*

Microarrays & Spot Colour*

Microarray Analysis Examples

Lung 20,224 Liver 37,807 Prostate 7,971 Skin 3,043 Brain 67,679 Heart 9,400 Colon 4,832

Brain

Lung

Liver
Bone 4,832

Liver Tumor

Yeast Two-Hybrid Analysis*

Yeast two-hybrid experiments yield information on protein protein interactions GAL4 Binding Domain GAL4 Activation Domain X and Y are two proteins of interest If X & Y interact then reporter gene is expressed

Example of 2-Hybrid Analysis

Uetz P. et al., A Comprehensive Analysis of Protein-Protein Interactions in Saccharomyces cerevisiae Nature 403:623-627 (2000)
High Throughput Yeast 2 Hybrid Analysis 957 putative interactions 1004 of 6000 predicted proteins involved

Example of 2-Hybrid Analysis

Rain JC. et al., The protein-protein interaction map of Helicobacter pylori Nature 409:211-215 (2001)
High Throughput Yeast 2 Hybrid Analysis 261 H. pylori proteins scanned against genome >1200 putative interactions identified Connects >45% of the H. pylori proteome

Another Way?
Ho Y, Gruhler A, et al. Systematic identification of protein complexes in Saccharomyces cerevisiae by mass spectrometry. Nature 415:180-183 (2002) High Throughput Mass Spectral Protein Complex Identification (HMS-PCI) 10% of yeast proteins used as bait 3617 associated proteins identified 3 fold higher sensitivity than yeast 2-hybrid

Affinity Pull-down*

Example of Affinity Pull-Down

Butland G, et al. Interaction network containing conserved and essential protein complexes in Escherichia coli Nature. 2005 Feb 3;433(7025):531-7 1000 proteins tagged 648 could be purified to homogeneity and their interacting protein partners identified by mass spectrometry

The E. coli Interaction Network

Proteomics Tools*
Molecular Biology Tools Separation & Display Tools Protein Identification Tools Protein Structure Tools

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Ciphergen Protein Chips*

Ciphergen Protein Chips

Hydrophobic (C8) Arrays Hydrophilic (SiO2) Arrays Anion exchange Arrays Cation exchange Arrays Immobilized Metal Affinity (NTA-nitroloacetic acid) Arrays Epoxy Surface (amine and thiol binding) Arrays

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Ciphergen Protein Chips*

Normal

Anaerobic

Protein Arrays

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Different Kinds of Protein Arrays*

Antibody Array Antigen Array Ligand Array

Detection by: SELDI MS, fluorescence, SPR, electrochemical, radioactivity, microcantelever

Protein (Antigen) Chips*

H Zhu, J Klemic, S Chang, P Bertone, A Casamayor, K Klemic, D Smith, M Gerstein, M Reed, & M Snyder (2000).Analysis of yeast protein kinases using protein chips. Nature Genetics 26: 283-289

ORF GST

His6

Nickel coating

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Protein (Antigen) Chips

Nickel coating

Arraying Process

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Probe with anti-GST Mab

Nickel coating

Anti-GST Probe

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Probe with Cy3-labeled Calmodulin

Nickel coating

Functional Protein Array*

Nickel coating

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Proteomics Tools*
Protein Identification Tools
Edman degradation Gel analyses MS fingerprinting and MS/MS

Protein Structure Tools

X-ray crystallography NMR spectroscopy

3 Kinds of Proteomics*
Expressional 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

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Expressional Proteomics

2-D Gel

QTOF Mass Spectrometry

Expressional Proteomics

+Arabinose

-Arabinose

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Expressional Proteomics

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

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Functional Proteomics*

Functional Proteomics (in silico)

AHGQSDFILDEADGMMKSTVPN HGFDSAAVLDEADHILQWERTY GGGNDEYIVDEADSVIASDFGH
*[LIVM][LIVM]DEAD*[LIVM][LIVM]*
(EIF 4A ATP DEPENDENT HELICASE)

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Functional Proteomics (in vitro)

Multi-well plate readers Full automation/robotics Fluorescent and/or chemiluminescent detection Small volumes (L) Up to 1536 wells/plate Up to 200,000 tests/day Mbytes of data/day

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

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Why Functional Proteomics?*

Concerned with the identification and classification of protein functions, activities 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

Examples
Edwards JS & Palsson BO Systems properties of the H. influenzae Rd metabolic genotype J. Biol. Chem. 274:17410-17416 (1999)
First example of metabolic/phenotypic prediction using proteome-wide information Converting sequence data to differential equations so as to predict biology/behavior

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From Genotype to Phenotype

Examples
Martzen MR, McCraith SM, Spinelli SL, Torres FM, Fields S, Grayhack EJ, Phizicky EM A biochemical genomics approach for identifying genes by the activity of their products Science 286:1153-1155 (1999)
Genome-wide Protein Tagging of 6144 ORFs Uses GST fusions and conventional assays

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Structural Proteomics*
High Throughput protein structure determination via Xray crystallography, NMR spectroscopy or comparative molecular modeling

Structural Proteomics: The Goal

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Structural Proteomics: The Motivation*

2000000 1800000 1600000 1400000 1200000 1000000 800000 600000 400000 200000 0 1980 1985 1990 1995 2000
200000 180000 160000 140000 120000 100000 80000 60000 40000 20000 0

Sequences

Structures Structures

2005

The Protein Fold Universe

How Big Is It???

500? 2000? 10000? ?

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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

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.

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Why Structural Proteomics?*

Structure Structure Function Mechanism

Structure-based Drug Design Solving the Protein Folding Problem

Keeps Structural Biologists Employed

Structural Proteomics - Status

18 registered centres (~30 organisms) 50330 targets have been selected 25202 targets have been cloned 14728 targets have been expressed 5122 targets are soluble 600 X-ray structures determined 164 NMR structures determined 633 Structures deposited in PDB

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Structural Proteomics - Status

135 structures deposited by Riken 117 structures deposited by Mid-West 85 structures deposited by North-East 74 structures deposited by New York 59 structures deposited by JCSG (UCSD) 34 structures deposited by Berkeley 24 structures deposited by Montreal/Kingston

Genomics, Proteomics & Systems Biology*

Genomics Proteomics

Systems Biology 1990 1995 2000 2005 2010 2015 2020

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