High Throughput Methods in Proteomics by ugg51907

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									   High Throughput Methods
         in Proteomics

        David Wishart
        University of Alberta
        Edmonton, AB
        david.wishart@ualberta.ca
Lecture 1.1                         1
               Proteomics
Proteomics employs an incredibly diverse
range of technologies including:
– molecular biology   – X-ray crystallography
– chromatography      – NMR spectroscopy
– electrophoresis     – microscopy
– mass spectrometry   – computational biology


 Lecture 1.1                               2
              Proteomics Tools

• Molecular Biology Tools
• Separation & Display Tools
• Protein Identification Tools
• Protein Structure Tools
Lecture 1.1                      3
       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
Lecture 1.1                                 4
                  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
Lecture 1.1                                 5
              SAGE Tools




Lecture 1.1                6
                        SAGE

Convert mRNA
to dsDNA


Digest with NlaIII



Split into 2 aliquots

Attach
Linkers



Lecture 1.1                    7
                    SAGE
Linkers have
PCR & Tagging
Endonuclease


Cut with TE
BsmF1

Mix both aliquots
Blunt-end ligate
to make “Ditag”

Concatenate
& Sequence

Lecture 1.1                8
SAGE of Yeast Chromosome




Lecture 1.1            9
              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
Lecture 1.1                                   10
              Gene Chip Tools




Lecture 1.1                     11
              DNA Microarrays




Lecture 1.1                     12
              DNA Microarray




Lecture 1.1                    13
   Microarrays & Spot Colour




Lecture 1.1                    14
  Microarray Analysis Examples
                  Brain
                  67,679
Lung                         Brain    Lung
20,224
                     Heart
                     9,400
Liver
37,807               Colon
                     4,832
Prostate
7,971
                             Liver   Liver Tumor
                     Bone
                     4,832
  Skin
  3,043


    Lecture 1.1                             15
              Microarray Software




Lecture 1.1                         16
  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

Lecture 1.1                           17
     Invitrogen Yeast 2-Hybrid
               X
              LexA
                          lacZ

                           B42
                          LexA

                         Y
                          lacZ

                         B42

               X     Y
              LexA
                          lacZ


Lecture 1.1                      18
 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

  Lecture 1.1                                19
 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

  Lecture 1.1                               20
                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
  Lecture 1.1                                     21
              Affinity Pull-down




Lecture 1.1                        22
       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
Lecture 1.1                                23
   Yeast Protein Localization




              Huh, K et al., Nature, 425:686-691(2003)
Lecture 1.1                                              24
     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
 Lecture 1.1                                25
   22 Different Cellular Zones




Lecture 1.1                      26
         GFP Tagging the Yeast
              Proteome




Lecture 1.1                      27
    Fluorescence Microscopy
               Nucleus   Nuclear Periphery   Endoplasmic Retic.




              Bud Neck   Mitochondria        Lipid particles




Lecture 1.1                                                       28
 Confirmation by Co-localization
       (GFP/RFP merging)




Lecture 1.1                   29
              Results




Lecture 1.1             30
              Proteomics Tools

• Molecular Biology Tools
• Separation & Display Tools
• Protein Identification Tools
• Protein Structure Tools
Lecture 1.1                      31
  Separation & Display Tools

• 1D Slab Gel Electrophoresis
• 2D Gel Electrophoresis
• Capillary Electrophoresis
• HPLC (SEC, IEC, RP, Affinity, etc.)
• Protein Chips
Lecture 1.1                        32
              SDS PAGE




Lecture 1.1              33
              SDS PAGE Tools




Lecture 1.1                    34
    Isoelectric Focusing (IEF)




Lecture 1.1                      35
              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


Lecture 1.1                              36
              2D Gel Principles
                     IEF




                                  SDS
                                  PAGE


Lecture 1.1                              37
 Advantages and Disadvantages
• Provides a hard-copy         • Limited pI range (4-8)
  record of separation         • Proteins >150 kD not
• Allows facile quantitation     seen in 2D gels
• Separation of up to 9000     • Difficult to see
  different proteins             membrane proteins
• Highly reproducible            (>30% of all proteins)
• Gives info on Mw, pI and     • Only detects high
  post-trans modifications       abundance proteins
• Inexpensive                    (top 30% typically)
                               • Time consuming
 Lecture 1.1                                         38
              2D Gel Software




Lecture 1.1                     39
     Capillary Electrophoresis




Lecture 1.1                      40
     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

Lecture 1.1                                41
               Chromatography
• Size Exclusion (size)
• Reverse Phase (hphob)
• Ion Exchange (charge)
• Normal Phase (TLC)
• Affinity (ligand)
• HIC (hydrophobicity)
• 2D Chromatography

 Lecture 1.1                    42
      Ciphergen Protein Chips




Lecture 1.1                     43
      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
Lecture 1.1                                  44
      Ciphergen Protein Chips
              Normal




              Tumor




Lecture 1.1                     45
              Protein Arrays




Lecture 1.1                    46
     Different Kinds of Protein
               Arrays
Antibody Array       Antigen Array       Ligand Array




    Detection by: SELDI MS, fluorescence, SPR,
    electrochemical, radioactivity, microcantelever
Lecture 1.1                                           47
          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
  Lecture 1.1                                                        48
        Protein (Antigen) Chips




              Nickel coating
Lecture 1.1                       49
              Arraying Process




Lecture 1.1                      50
      Probe with anti-GST Mab




              Nickel coating
Lecture 1.1                     51
              Anti-GST Probe




Lecture 1.1                    52
        Probe with Cy3-labeled
             Calmodulin




              Nickel coating
Lecture 1.1                      53
   “Functional” Protein Array




Nickel coating

Lecture 1.1                     54
              Proteomics Tools

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

Lecture 1.1                       55
              Microsequencing




                 Electro-blotting

Lecture 1.1                         56
              Edman Sequencing




Lecture 1.1                      57
              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
Lecture 1.1                              58
 Protein ID by MS and 2D gel




Lecture 1.1                59
 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

Lecture 1.1                                    60
              Protein ID Protocol




Lecture 1.1                         61
              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

Lecture 1.1                     62
              MS Analysis Software




                       Protein Prospector
                       MS-Fit
                       Mowse
                       PeptideSearch
                       PROWL

Lecture 1.1                                 63
              Proteomics Tools

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

Lecture 1.1                      64
   Protein Structure Initiative

•35,000 proteins

•10,000 subset

•30% ID or

•30 seq                    30 seq
•Solve by 2010

•$20,000/Structure

Lecture 1.1                       65
       Structure Determination




              NMR      X-ray
Lecture 1.1                      66
              X-ray Crystallography


                       FT




Lecture 1.1                           67
              NMR Spectroscopy



                     FT




Lecture 1.1                      68
       Structure Determination




Lecture 1.1                      69
                   Bottlenecks
           X-ray                       NMR
• Producing enough             • Producing enough
  protein for trials             labeled protein for
• Crystallization time and       collection
  effort                       • Sample “conditioning”
• Crystal quality, stability   • Size of protein
  and size control             • Assignment process is
• Finding isomorphous            slow and error prone
  derivatives                  • Measuring NOE’s is
• Chain tracing & checking       slow and error prone

 Lecture 1.1                                        70
              Protein Expression




Lecture 1.1                        71
         Robotic Crystallization




Lecture 1.1                        72
    Synchrotron Light Source




Lecture 1.1                    73
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
Lecture 1.1                            74
          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)

Lecture 1.1                                 75
              Automated Structure
                  Generation




Lecture 1.1                         76
NMR & Structural Proteomics




Lecture 1.1                                                         77
              Proc. Natl. Acad. Sci. USA, Vol. 99,1825-1830, 2002
NMR & Structural Proteomics




Lecture 1.1                                                         78
              Proc. Natl. Acad. Sci. USA, Vol. 99,1825-1830, 2002
 Auto-comparative Modeling




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     ASDEYAHLRILDPQRSTVAYAYE--KSFAPPGSFKWEYEAHADS
     MCDEYAHIRLMNPERSTVAGGHQWERT----GSFKEWYAAHADD


Lecture 1.1                                         79
              The Goal




Lecture 1.1              80

								
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