Proteomics

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Proteomics

• Micro 343
• David Wishart Rm. Ath 3-41
• david.wishart_at_ualberta.ca

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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

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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).

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Proteomics
Proteomics employs an incredibly diverse range of
technologies including

• molecular biology
• chromatography
• electrophoresis
• mass spectrometry

• X-ray crystallography
• NMR spectroscopy
• robotics
• computational biology

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Proteomics Tools

• Molecular Biology Tools
• Separation Display Tools
• Protein Identification Tools
• Protein Structure Tools

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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

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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

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Gene Chip Tools
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DNA Microarrays
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DNA Microarray
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Microarrays Spot Colour
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Microarray Analysis Examples
Brain
Lung
Liver
Liver Tumor
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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

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Example of 2-Hybrid Analysis

• Uetz P. et al., A Comprehensive Analysis of
  Protein-Protein Interactions in Saccharomyces
  cerevisiae Nature 403623-627 (2000)
• High Throughput Yeast 2 Hybrid Analysis
• 957 putative interactions
• 1004 of 6000 predicted proteins involved

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Example of 2-Hybrid Analysis

• Rain JC. et al., The protein-protein interaction
  map of Helicobacter pylori Nature 409211-215
  (2001)
• High Throughput Yeast 2 Hybrid Analysis
• 261 H. pylori proteins scanned against genome
• gt1200 putative interactions identified
• Connects gt45 of the H. pylori proteome

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Another Way?

• Ho Y, Gruhler A, et al. Systematic identification
  of protein complexes in Saccharomyces cerevisiae
  by mass spectrometry. Nature 415180-183 (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

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Affinity Pull-down
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Example of Affinity Pull-Down

• Butland G, et al. Interaction network containing
  conserved and essential protein complexes in
  Escherichia coliNature. 2005 Feb
  3433(7025)531-7
• 1000 proteins tagged
• 648 could be purified to homogeneity and their
  interacting protein partners identified by mass
  spectrometry

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The E. coli Interaction Network
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Proteomics Tools

• Molecular Biology Tools
• Separation Display Tools
• Protein Identification Tools
• Protein Structure Tools

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Ciphergen Protein Chips
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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
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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
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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
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Arraying Process
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Probe with anti-GST Mab
Nickel coating
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Anti-GST Probe
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Probe with Cy3-labeled Calmodulin
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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

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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
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Expressional Proteomics
Arabinose -Arabinose
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Expressional Proteomics
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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
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Functional Proteomics (in silico)
AHGQSDFILDEADGMMKSTVPN HGFDSAAVLDEADHILQWERTY
GGGNDEYIVDEADSVIASDFGH LIVMLIVMDEADLIVM
LIVM (EIF 4A ATP DEPENDENT HELICASE)
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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

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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

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Examples

• Edwards JS Palsson BO Systems properties of
  the H. influenzae Rd metabolic genotype J. Biol.
  Chem. 27417410-17416 (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
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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 2861153-1155
  (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 X-ray crystallography, NMR spectroscopy or
  comparative molecular modeling

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Structural ProteomicsThe Goal
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Structural Proteomics The Motivation
200000
180000
160000
140000
120000
100000
Sequences
Structures
80000
60000
40000
20000
0
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The Protein Fold Universe
500? 2000? 10000?
How Big Is It???
8
?
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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

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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 Function
• Structure Mechanism
• Structure-based Drug Design
• Solving the Protein Folding Problem
• Keeps Structural Biologists Employed

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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

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Genomics, Proteomics Systems Biology
Genomics
Proteomics
Systems Biology