Introduction to Proteomics

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Proteomics
Session 13 Other applications in current
proteomics
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Applications of Proteomics

• 1. Protein Complexes Mining
• 2. Yeast Two-hybrid system (in vivo PIP)
• 3. Phage display and cell surface display system
  (in vitro PIP)
• 4. Protein Arrays
• 5. SELDI protein chips (Ciphergen)
• 6. Multi-dimensional HPLC (MDLC)

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1. Protein Complexes Mining
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1. Proteome Complex Mining

• A functional proteomics approach
• A Proteome Mine Example
• ATP is immobilized to beads in protein kinase
  conformation
• Total protein is mixed the beads and the mixture
  washed
• Remaining proteins isolated and
  identifiedprotein kinases, and purine dependent
  metabolic enzymes
• Immobilize a putative drug to bead and test for a
  cellular ligand

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Cell mapping Protein and Protein interaction
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Enriching methods

• Coimmunoprecipitation (Adams et al. 2002, eg.
  Anti p53 antibody)
• Coprecipitation (Seraphin et al. 2003, eg. V5
  epitope)
• Protein affinity-interaction chromatography
  (Einarson and Orlinick 2002, eg. GST fusion
  protein)
• Isolation of intact multiprotein complexes (eg.
  Nuclear pore complexes, ribosome complexes, and
  spliceosomies)

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Representative macromolecular complexes
identified by proteomics methods
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2. Yeast Two-hybrid system (in vivo PIP)
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2. Yeast Two-Hybrid System (in vivo)

• Interaction of bait and prey proteins localizes
  the activation domain to the reporter gene, thus
  activating transcription.
• Since the reporter gene typically codes for a
  survival factor, yeast colonies will grow only
  when an interaction occurs.

Activation Domain
Prey Protein
Reporter mRNA
Bait Protein
Reporter mRNA
Reporter mRNA
Reporter mRNA
Binding Domain
Reporter mRNA
Reporter Gene
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Yeast 2 hybrid system, contd.
X
Y1, Y2, Y3, Y5, Y6(all genome)
X
Y4
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More complexed Yeast 2 hybrid system
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3. Phage display and cell surface display system
(in vitro PIP)
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3. Phage display system (in vitro)
Biopanning
Phage minor coat protein
SCIENCE VOL 298 18 OCTOBER 2002
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Applications for Phage display system
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Cell surface display

• Fuse the gene of expressed proteins to the genes
  of anchor proteins of host.
• Express protein on the surface of host (bacteria
  or yeast)
• Screen the positive clones by FACS

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Compare with Phage Display

• Phage Display
• Size limitation insert DNA is smaller
• No PTM
• Can not express larger proteins
• Cell Surface display
• Via eukaryote or prokaryote
• Express larger proteins with PTM

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Bacteria Cell Surface Display

• Gram-negative bacteria is commonly used.
• Normally translated
• Transported to membrane
• Anchored on the outer membrane and face to
  extracellularly
• Membrane protein of E.ColiLamB?OmpA?OmpC?OmpS?Omp
  T?FhuA ? the lipoprotein TraT

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Lpp-OmpA system Bacteria Cell Surface Display

• Anchor protein Lpp-OmpA hybrid
• Lipoprotein (Lpp) outer membrane localization
  domain fused to amino acids 46-159 of outer
  membrane protein A (OmpA)
• C-terminal of anchor protein fused with
  recombinant polypeptide
• First successful application.
• cellulases?esterase?organophosphate
  hydrolases?scFv enzymes?b-lactamase?thioredoxin

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Yeast Cell Surface Display

• Stablize expressed protein with thick cell Wall.
• Similar protein secretion, post-translational
  modification with eukaryotes
• Suitable for express antibody fragments,
  cytokines and receptor extra cellular domains
• Saccharomyces cerevisiae
• Hansenula polymorpha

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Two types of east Cell Surface Display

1. Glycosylphosphatidyl-inositol (GPI) -anchored
   cell surface proteins
2. Yeast-surface receptor a-agglutinin

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GPI-anchored proteins

• Cell surface proteins in all eukaryotic cells
• 5104 molecules in the surface of S. cerevisiae
• Link target proteins via C-terminal
• HpCWP1 in H. polymorpha (Kim et al., 2002)

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GPI-anchored proteins
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Yeast-surface receptor a-agglutinin

• AGa1 (S. cerevisiae)
• In a mating type
• Highly glycosylated cell wall anchored proteins
• 650 amino acids with secretion signal at
  N-termanal and GPI at C-terminal
• Express recombinant proteins extracellularly.

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4. Protein Arrays
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4. Protein (micro) arrays

• Another Functional Proteomics Approach
• Same concept as a DNA Array
• Has been published in a peer-reviewed journal
• Too much expectation lies in with.

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Technological Components for Protein Chips
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Protein Microarrays
Science, 289, 1760, 2000

• Microspotting of proteins on aldehyde glass
  slide
• 150200µm in diameter (100 µg/mL)
• 10,799 spots of Protein G (1,600 spots/cm2)
• A single spot of FRB (FKBP12-rapamycin binding)

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Protein MicroarrayG. MacBeath and S.L.
Schreiber, 2000, Science 2891760
Spotting platform and protein microarray
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What protein microarray can do?

1. Protein / protein interaction
2. Enzyme / substrate interaction (transient)
3. Protein / small molecule interaction
4. Protein / lipid interaction
5. Protein / glycan interaction
6. Protein / Ab interaction

Reference 1. G. MacBeath and S.L. Schreiber,
2000, Science 2891760 2. H.Zhu et al, 2001
Science 2932101 3. Ziauddin J and Sabatini DM,
2001 Nature 411107
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Application of protein microarray
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Protein microarrays (Ab arrays)
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Face the real world
The true spot quality from real experiment
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Class of capture molecule for protein microarray
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Core Technologies in Protein Chip
Protein chips for practical use
Detecting the biomolecular interaction with high
sensitivity and reliability
How to construct the monolayers of biomolecules
on a solid surface
Most difficult parts

• Maximizing the binding efficiency
• Maximizing the fraction of active biomolecules
• Minimizing the nonspecific protein binding

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5. SELDI protein chips (Ciphergen)
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5. SELDI protein chip (Ciphergen)

• SELDI surface enhanced laser desorption/
  ionization

Protein chips
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Types of protein chip
IMAC30?immobilized metal affinity capture array
with a nitriloacetic acid (NTA) surface with an
updated hydrophobic barrier coating. IMAC3?mmobili
zed metal affinity capture array with a
nitriloacetic acid (NTA) surface. CM10?weak
cation exchange array with carboxylate
functionality, with an updated hydrophobic
barrier coating WCX2?weak cation exchange array
with carboxylate functionality. Q10?strong anion
exchange array with quaternary amine
functionality, with an updated hydrophobic
barrier coating. SAX2?strong anion exchange
array with quaternary amine functionality.
H50?bind proteins through reversed phase or
hydrophobic interaction chromatography with an
updated hydrophobic barrier coating H4 mimic
reversed phase chromatography with C16
functionality. NP20?mimic normal phase
chromatography with silicate functionality
Au?old chips to be used directly for MALDI-based
experiments
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SELDI protein chip (Ciphergen), contd.
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SELDI protein chip (Ciphergen), application
Representative raw spectra and gel-view
(grey-scale) of serum from a normal donor, and
from patients with either BPH (benign prostate
hyperplasia) or prostate cancer (PCA) using the
IMAC3-Cu chip chemistry (Virginia Prostate
Center).
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6. Multi-dimensional HPLC (MDLC)
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Configuration of MDLC
2nd RP
1st SCX
From N341 Institute of Biomedical Sciences ,
Academia Sinica
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Result from SCX and 2D LC
From N341 Institute of Biomedical Sciences ,
Academia Sinica
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Agilent 1100 series
MASS
MDLC