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Proteomics

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957 putative interactions. 1004 of 6000 predicted proteins involved. Example of 2-Hybrid Analysis ... 1200 putative interactions identified. Connects 45% of ... – PowerPoint PPT presentation

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Title: Proteomics


1
Proteomics
  • Micro 343
  • David Wishart Rm. Ath 3-41
  • david.wishart_at_ualberta.ca

2
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

3
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).

4
Proteomics
Proteomics employs an incredibly diverse range of
technologies including
  • molecular biology
  • chromatography
  • electrophoresis
  • mass spectrometry
  • X-ray crystallography
  • NMR spectroscopy
  • robotics
  • computational biology

5
Proteomics Tools
  • Molecular Biology Tools
  • Separation Display Tools
  • Protein Identification Tools
  • Protein Structure Tools

6
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

7
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

8
Gene Chip Tools
9
DNA Microarrays
10
DNA Microarray
11
Microarrays Spot Colour
12
Microarray Analysis Examples
Brain
Lung
Liver
Liver Tumor
13
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

14
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

15
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

16
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

17
Affinity Pull-down
18
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

19
The E. coli Interaction Network
20
Proteomics Tools
  • Molecular Biology Tools
  • Separation Display Tools
  • Protein Identification Tools
  • Protein Structure Tools

21
Ciphergen Protein Chips
22
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

23
Ciphergen Protein Chips
Normal
Anaerobic
24
Protein Arrays
25
Different Kinds of Protein Arrays
Antibody Array Antigen Array
Ligand Array
Detection by SELDI MS, fluorescence, SPR,
electrochemical, radioactivity, microcantelever
26
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
27
Protein (Antigen) Chips
Nickel coating
28
Arraying Process
29
Probe with anti-GST Mab
Nickel coating
30
Anti-GST Probe
31
Probe with Cy3-labeled Calmodulin
32
Functional Protein Array
Nickel coating
33
Proteomics Tools
  • Protein Identification Tools
  • Edman degradation
  • Gel analyses
  • MS fingerprinting and MS/MS
  • Protein Structure Tools
  • X-ray crystallography
  • NMR spectroscopy

34
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

35
Expressional Proteomics
2-D Gel QTOF Mass Spectrometry
36
Expressional Proteomics
Arabinose -Arabinose
37
Expressional Proteomics
38
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

39
Functional Proteomics
40
Functional Proteomics (in silico)
AHGQSDFILDEADGMMKSTVPN HGFDSAAVLDEADHILQWERTY
GGGNDEYIVDEADSVIASDFGH LIVMLIVMDEADLIVM
LIVM (EIF 4A ATP DEPENDENT HELICASE)
41
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

42
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

43
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

44
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

45
From Genotype to Phenotype
46
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

47
Structural Proteomics
  • High Throughput protein structure determination
    via X-ray crystallography, NMR spectroscopy or
    comparative molecular modeling

48
Structural ProteomicsThe Goal
49
Structural Proteomics The Motivation
200000
180000
160000
140000
120000
100000
Sequences
Structures
80000
60000
40000
20000
0
50
The Protein Fold Universe
500? 2000? 10000?
How Big Is It???
8
?
51
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

52
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.

53
Why Structural Proteomics?
  • Structure Function
  • Structure Mechanism
  • Structure-based Drug Design
  • Solving the Protein Folding Problem
  • Keeps Structural Biologists Employed

54
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

55
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

56
Genomics, Proteomics Systems Biology
Genomics
Proteomics
Systems Biology
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