High Throughput Methods in Proteomics

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High Throughput Methods in Proteomics

• David Wishart
• University of Alberta
• Edmonton, AB
• david.wishart_at_ualberta.ca

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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
• microscopy
• 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
• GFP Tagging Microscopy

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SAGE

• Principle is to convert every mRNA molecule into
  a short (10-14 base), unique tag. Equivalent to
  reducing all the people in a city into a
  telephone book with surnames
• After creating the tags, these are assembled or
  concatenated into a long list
• The list can be read using a DNA sequencer and
  the list compared to a database to ID genes or
  proteins and their frequency

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SAGE Tools
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SAGE
Convert mRNA to dsDNA Digest with
NlaIII Split into 2 aliquots Attach Linkers
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SAGE
Linkers have PCR Tagging Endonuclease Cut
with TE BsmF1 Mix both aliquots Blunt-end
ligate to make Ditag Concatenate Sequence
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SAGE of Yeast Chromosome
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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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Microarray Software
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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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Invitrogen Yeast 2-Hybrid
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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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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
• GFP Tagging Microscopy

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Yeast Protein Localization
Huh, K et al., Nature, 425686-691(2003)
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Yeast Proteome Localized

• Used 6234 yeast strains expressing full-length,
  chromosomally tagged green fluorescent protein
  (GFP) fusion proteins
• Measured localization by fluorescence microscopy
• Localized 75 of the yeast proteome, into 22
  distinct subcellular localization categories
• Provided localization information for 70 of
  previously unlocalized proteins

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22 Different Cellular Zones
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GFP Tagging the Yeast Proteome
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Fluorescence Microscopy
Nucleus Nuclear Periphery
Endoplasmic Retic.
Bud Neck Mitochondria
Lipid particles
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Confirmation by Co-localization (GFP/RFP merging)
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Results
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Proteomics Tools

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

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Separation Display Tools

• 1D Slab Gel Electrophoresis
• 2D Gel Electrophoresis
• Capillary Electrophoresis
• HPLC (SEC, IEC, RP, Affinity, etc.)
• Protein Chips

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SDS PAGE
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SDS PAGE Tools
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Isoelectric Focusing (IEF)
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Isoelectric Focusing

• Separation of basis of pI, not Mw
• Requires much higher voltages
• Requires much longer period of time
• IPG (Immobilized pH Gradient)
• Typically done in strips or tubes (to facilitate
  2D gel work)
• Uses ampholytes to establish pH gradient

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2D Gel Principles
IEF
SDS PAGE
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Advantages and Disadvantages

• Provides a hard-copy record of separation
• Allows facile quantitation
• Separation of up to 9000 different proteins
• Highly reproducible
• Gives info on Mw, pI and post-trans modifications
• Inexpensive

• Limited pI range (4-8)
• Proteins gt150 kD not seen in 2D gels
• Difficult to see membrane proteins (gt30 of all
  proteins)
• Only detects high abundance proteins (top 30
  typically)
• Time consuming

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2D Gel Software
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Capillary Electrophoresis
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Capillary Electrophoresis

• Capillary Zone Electrophoresis (CZE)
• Separates on basis of m/z ratio
• Capillary Gel Electrophoresis (CGE)
• Separates by MW and m/z ratio
• Capillary Isoelectric Focusing (CIEF)
• Separates on basis of pI
• 2-Dimensional Electrophoresis (2D-CE)
• Separates using tandem CE methods

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Chromatography

• Size Exclusion (size)
• Reverse Phase (hphob)
• Ion Exchange (charge)
• Normal Phase (TLC)
• Affinity (ligand)
• HIC (hydrophobicity)
• 2D Chromatography

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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
Tumor
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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

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

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Microsequencing
Electro-blotting
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Edman Sequencing
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Microsequencing

• Generates sequence info from N terminus
• Commonly done on low picomolar amounts of protein
  (5-50 ng)
• Newer techniques allow sequencing at the
  femtomolar level (100 pg)
• Up to 20 residues can be read
• Allows unambiguous protein ID for 8 AA
• Relatively slow, modestly expensive

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Protein ID by MS and 2D gel
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Protein ID by MS and 2D gel

• Requires gel spots to be cut out (tedious)
• Ideal for high throughput (up to 500 samples per
  day)
• Allows modifications to be detected
• MS allows protein identification by
• Intact protein molecular weight
• Peptide fingerprint molecular weights
• Sequencing through MS/MS

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Protein ID Protocol
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Typical Results

• 401 spots identified
• 279 gene products
• Confirmed by SAGE, Northern or Southern
• Confirmed by amino acid composition
• Confirmed by amino acid sequencing
• Confirmed by MW pI

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MS Analysis Software
Protein Prospector MS-Fit Mowse PeptideSearch PROW
L
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Proteomics Tools
Molecular Biology Tools Separation Display
Tools Protein Identification Tools Protein
Structure Tools
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Protein Structure Initiative

• 35,000 proteins
• 10,000 subset
• 30 ID or
• 30 seq
• Solve by 2010
• 20,000/Structure

30 seq
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Structure Determination
NMR X-ray
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X-ray Crystallography
F T
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NMR Spectroscopy
F T
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Structure Determination
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Bottlenecks
X-ray NMR

• Producing enough protein for trials
• Crystallization time and effort
• Crystal quality, stability and size control
• Finding isomorphous derivatives
• Chain tracing checking

• Producing enough labeled protein for collection
• Sample conditioning
• Size of protein
• Assignment process is slow and error prone
• Measuring NOEs is slow and error prone

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Protein Expression
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Robotic Crystallization
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Synchrotron Light Source
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MAD X-ray Crystallography

• MAD (Multiwavelength Anomalous Dispersion
• Requires synchrotron beam lines
• Requires protein with multiple scattering centres
  (selenomethionine labeled)
• Allows rapid phasing
• Proteins can now be solved in just 1-2 days

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High Throughput NMR

• Higher magnetic fields (From 400 MHz to 900 MHz)
• Higher dimensionality (From 2D to 3D to 4D)
• New pulse sequences (TROSY, CBCANNH)
• Improved sensitivity
• New parameters (Dipolar coupling, cross
  relaxation)

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Automated Structure Generation
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NMR Structural Proteomics
Proc. Natl. Acad. Sci. USA, Vol. 99,1825-1830,
2002
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NMR Structural Proteomics
Proc. Natl. Acad. Sci. USA, Vol. 99,1825-1830,
2002
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Auto-comparative Modeling
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AHLRILDPQRSTVAYAYE--KSFAPPGSFKWEYEAHADS MCDEYAHIRL
MNPERSTVAGGHQWERT----GSFKEWYAAHADD
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The Goal