Protein Chip??? ?? ??? ? Ligand? microarray ??

1
NanoBiotechnology and Protein Chips
? ? ? KAIST ?????
2
Molecularly Organized Nanostructures

• Analytical Sciences
• Nano-pore ( Bacterial pore-forming protein
  alpha-Hemolysin)
• for single molecule analysis
• Molecular Devices
• Nano-device, Nano-wire, Molecular
  computation
• Biotechnology
• Bioelectronic devices
• Biosensors Biochips ( DNA Protein arrays
  )
• Biomolecules Proteins, DNA, Peptides, Ligands,
  Antibody-Antigen, Receptors

3
(No Transcript)
4
Detection of a variety of analytes by stochastic
sensing
5
Lipid bilayer arrays
6
Binding of cyclodextrin (CD) and s7 CD to PNQ
7
Binding of guest molecules G1 and G2 to PNQ
CDs7 CD
8
Capture of a single small molecule within the
nanocavity of PNQ CDs7 CD
9
Carbon Nanotube Nanoelectrode Array for
Ultrasensitive DNA Detection
10
(No Transcript)
11
(No Transcript)
12
Electronic Detection of Specific Protein Binding
Using Nanotube FET Devices
13
(No Transcript)
14
(No Transcript)
15
(No Transcript)
16
Unzipping Dna Oligomers
17
(No Transcript)
18
(No Transcript)
19
Tasks in the Post-Genomic Era To understand the
functions, modifications, and regulations of
every encoded proteins in cells
Analysis of protein interactions in parallel with
high speed
20
Chip-Based Technologies
DNA Chip Genome-wide expression profiling
Drug discovery Diagnostics Understanding living
systems
Identification of
protein function
Protein Chip Gene expression, Biomolecular
functions, Cell signaling networks
21
Applications of Protein Chip
Disease Diagnosis Identification of disease
marker protein (Protein expression
profiles) Detection of disease marker protein
Protein Functions Characterization of biochemical
properties Protein folding and stability Expressio
n profiles Post-translational modification Proteol
ysis for activation Interactions with
biomolecules (proteins, ligands, DNA)
Drug Screening Identification of target
proteins Screening of bioactive ligands binding
to disease-associated proteins
Protein Function Analysis Protein-protein,
Protein-DNA, Protein-ligand, Antigen-antibody,
Receptor-ligand
22
Genome-Wide Prediction of Protein Function
23
Identification of Protein Functions/Interactions

• ? Characterization of biochemical properties
• ? Interactions with biomolecules
• (proteins, ligand, DNA)
• ? Protein folding and stability
• ? Expression profiles
• Post-translational modifications
• (phosphorylation or glycosylation)
• ? Proteolysis for activation

24
High Throughput Screening
" You get what you screen for"
Design of screening strategy
25
Drug Discovery Chemical Genomics
? Identifying the protein targets of small
molecules (synthesis or natural
compounds) ? Screening the small molecule for a
target protein
26
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)

27
BODIPY-IgG
Cy3IkBa
Cy5-FKBP Rapamycin ()
AP1497
Cy5-FKBP Rapamycin (-)
28
Target Proteins of Small Molecules
29
Analysis of Yeast Protein Kinases
Yeast genome 6,200 ORFs ? 122 Protein kinases ?
Ser/Thr family ? Tyr family ? Poly ( Tyr-Glu)

• Experimental Procedures
• ? Expression and purification of 119 kinases in
  GST-fusion proteins in E. coli
• Protein chips using silicone elastomer
• (PDMS poly(dimethylsiloxane)
• 10?14 rectangular array
• Attachment of 17 substrates
• ? High-throughput assay by phosphoimager
• 33P?-ATP and protein kinase
• (8?10-9?g/?m2 in each spot)

Nature Genetics, 26, 283-289, 2000
30
Kinase Substrates
? Kinase themselves( autophosphorylation) ?
Bovine histone H1 ? Bovine casein ? Myelin basic
protein ? Ax12 Carboxy-terminus GST ? Rad 9 ( a
phospho-protein involved in the DNA damage
checkpoint) ? Gic2 ( involved in budding) ? Red1
( involved in chromosome synapsis) ? Mek1 (
involved in chromosome synapsis) ? Poly
(Tyr-Glu) ? Ptk2 ( transport protein) ? Hsl1 (
involved in cell cycle regulation) ? Swi6 (
involved in G1/S control) ? Tub4 ( involved in
microtuble nucleation) ? Hog1 ( involved in
osmoregulation)
31
Global Analysis of Yeast Proteom by Protein Chips

• Proteom arrays with 5,800 ORFs from Yeast
• Major hurdle in proteom analysis Cloning and
  expression of clones and purification of proteins
  in a high throughput fashion
• GST-HisX6-fused proteins -? Proteom arrays on
  nickel-coated slide

Identification of new calmodulin and
phospholipid-interacting proteins
Snyder et al., Science, 26 July, 2001
32

• Immunoblot analysis of purified proteins using
  anti-GST antibody
• Proving of 5800 proteins with Cy5 labeled
  anti-GST antibody

33
Proving with anti-GST antibody labeled with Cy5
Proving with biotinylated calmodulin Detection
using Cy3-labeled streptavidin
Proving with biotinylated phosphoinositide(PI) Det
ection using Cy3-labeled streptavidin
PI second-messenger regulating the cellular
process like growth, differentiation, and
cytoskeletal rearrangement
Putative calmodulin binding motifs of 14
positive proteins Consequence is (I/L)QXK(K/X)GB
34
(No Transcript)
35
Profiling of Cancer Cells Using Protein
Microarrays

• Antibody arrays on poly-L-lysine coated or
  aldehyde-modified glass slides by robotic
  arrayer.
• Proteins were extracted from reference and test
  samples and labeled with either Cy5 or Cy3 dyes.
• The ratio, Cy5/Cy3, can be interpreted as
  relative protein abundance.

Sreekumar et al., Cancer Res. 61, 7585 (2001)
36
Profiling of Cancer Cells Using Protein
Microarrays

• 146 antibodies against proteins involved in
  stress response, cell cycle progression, and
  apoptosis
• Antibody arrays on PLL- or aldehyde coated
  slide 1920 element protein microarray consisting
  of 146 antibodies.
• ? Untreated LoVo cells (Cy3 labeled) vs Radiation
  treated LoVo cells (Cy5 labeled).

? Radiation-induced up-regulation of apoptotic
regulators and down-regulation of
carcinoembryonic antigen as observed.
37
Peptide Chip
? Immobilization of the kinase substrate
AclYGEFKKKC-NH2 by the Diels-Alder reaction
between the diene and quinone groups on a mixed
SAM ? Phosphorylation of the substrates by c-Src
kinase
? Arrays by commercial spotting robot ?
Detection of substrate phosphorylation by
immunofluorescence microscopy using
anti-phosphotyrosine antibody.
Houseman et al., Nat. Biotechnol. 20, 270 (2002)
38
Peptide Chip

• ? Quantitative evaluation of kinase inhibitors in
  array format
• Detecting the inhibition of phosporylation by
  inhibitor using phospho-image analyzer or
  anti-phosphotyrosin antibody
• ? Peptide arrays can be applicable to the
  identification of protein-ligand complexes,
  screening of specific inhibitor, or evaluation
  of kinase activities.

39
Dip-Pen Nanolithography

• Absorption of Proteins on preformed MHA
  patterns
• Characterization of the resulting protein arrays
  by AFM
• 100350nm features

Lee et al., Science 295, 1702 (2002)
40
Challenging Issues

• Feasibility tests with known proteins
• Development of viable protein chips

41

• Technology Platforms for Protein Chips

42
Protein-based Chips

• Defined binding site
• Protein levels factor of 10 6
• ( mRNA levels factor of 10 4)
• Difficulty in signal amplification
• Fragile

43
Technological Components for Protein Chips
44
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

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

45
Interfacing Material for the Biomolecular
Monolayers
With no interfaces Aggregation (dirty
island) Loss of biological functions Dissociation
With interfaces Retaining the biological
functions Solve the Orientation / NSB problems
46
Dendrimers Highly Branched Dendritic
Macromolecules
47
Poly (amido amine) Dendrimers

• Characteristics
• Monodisperse macromolecule
• Globular (Spherical)
• Facile surface bio-functionalization
• Similar molecular size to biomolecules
• (Glucose oxidase 6?5.2?7.7 nm)

• Applications

Vehicles for delivery of genes and drugs
4.5 nm G4 Poly(amidoamine) Dendrimer
Biomimetic catalysts (Peptides-, Glycodendrimers)
Medical applications (MRI contrast enhancer)
Molecular carriers for chemical catalysts (Core,
Peripheral)
48
Dendrimer Monolayers Fabrication and
Characterization

• SPR (surface plasmon resonance)
• spectroscopy
• Estimation of mass change on the chip
• surface

49
Formation of the Dendrimer Monolayers on Gold

• Quantitative analysis using SPR
• M Maximum mass of dendrimers that can be
  immobilized in a defined area
• Fractional surface coverage
• by immobilized dendrimers ?M / M
• Angle shift 0.34 degree
• Mass change 3.4 ng/cm2
• Surface fractional coverage 89

50
Applications of the Dendrimer Monolayer

• Monolayers of biomolecules in a spatially ordered
  manner
• Dendrimer as the interfacing material
• Microarraying of biomolecules in the molecularly
  organized manner

Platform or interface for immobilizing and
microarraying biomolecules
51
Streptavidin

• Characteristics
• Highest association constant with biotin ( 10
  15 M-1)
• Homo-tetramer with four binding sites for biotin
  at opposite direction
• Use of (strept)avidin-biotin couple
• Readily available wide variety of biotinylated
  species
• Sub-structure for the construction of
  supramolecular systems

52
Streptavidin Monolayers

• Model study of biomolecular interaction using
  avidin/biotin couple
• Application of avidin monolayers as biospecific
  platform
• for immobilization of the biotinylated
  biomolecules (Ka1015 M-1)

53
Avidin Platform on the Dendrimer Monolayer

• Synthetic Protocol
• Cleaned gold surface
• MUA SAM formation
• PF5 activation of MUA carboxyls
• PAMAM dendrimer monolayer formation
• Functionalization with amidocaproate- biotin(Ka
  1?1015 M-1, Extended form of biotin, known as a
  most stable biorecognition couple)
• Fluorophore-labeled Avidin
• Tracing of biospecific interaction
• Electrochemical test (Cyclic Voltammetry)
• Fluorescence / Confocal Microscopy
• SPR (Surface Plasmon Resonance)

54
Electrochemical Tracing of Avidin Coverage

• Double (Fc and Biotin) - functionalized
  dendrimer monolayer
• Signaling depending on the steric blockage by
  avidin
• GOx as a signal generator Catalytic signal
  amplification

55
Electrochemical Tracing of Avidin Coverage

• Experimental Condition for Cyclic Voltammetry
• Without Avidin binding reaction.
• After reaction with Avidin, 10?g/mL for 30min.
• GOx (0.22 ?M), Glucose (10 mM) substrate, and
  potential scan rate 5mV/s.

56
Fluorescence images of avidin monolayer
57
Quantification of the Bound Avidin Using SPR
SPR measurement with Biacore-X Functionalization
with NHS-sulfo-biotin to preformed dendrimer
monolayer PBST washing Reaction with avidin
sample (A) (50µg/mL, in PBST) PBST washing
(B) Recoding SPR response in a steady-state Inset
nonspecific binding of avidin
Surface Coverage of avidin, 5ng/mm2 90
coverage of gold surface
58
Comparison with Other Interfacing Materials
Interfacing materials having number of amine
groups, but different surface configuration
Dendrimer monolayer (64 amine groups)
Poly(L-lysine) (ca. 104)
Cystamine SAM
59
SPR Angle Shifts for Specific Binding of Avidin
60
Specific Coverage of Avidin vs. Surface Biotin
Density
JACS. 1999, 121, 6469 Langmuir, 2000, 16,
9421
61
Surface Configuration of Biotin-functionalized
Dendrimer Monolayers
62
Monolayers of Biomolecules on the Dendrimer
Monolayers

• Antibody
• General Protein
• Controlling the Orientation of Biomolecules

63
Antibody Layers on the Dendrimer Monolayers

• Structure Features

• Highest association constant with Ag
• (10 -8 10 -12 M-1)
• Two binding sites (Fv) for Ag

• Flexible polypeptides ? Segmental flexibility
• for effective Ag binding
• Fc region ? protein A affinity
• Hinge region ? Carbohydrate-rich

64
Model Antibodies
Chick Immunoglobulin G

• The same topology to antibody model
• Study for Ab-Ag interaction on the chip surface
• Performance test of the chip platform
• Orientation of protein probes
• Minimizing nonspecific protein
  binding

Antigen

• Antibody

Ferritin
Stills disease acute systemic inflammatory
disorder characterized by a triad of spiking
fever, skin rash and polyarthritis.

• A diagnostic marker of iron deficiency, Stills
  disease
• Elevated level in patients with chronic liver
  disease,
• lymphoid malignancy leukemia and Hodgkin
  lymphoma
• Associated with elevated risk of myocardial
  infarction

65
Controlling the Orientation of Antibody Molecules

• Strategies
• Maximizing the binding efficiency with antigen
• Antigen binding sites ( variable regions) should
  be exposed

66
Ab-Ag Interaction on Various Antibody Layers
67
Minimizing the Non-specific Protein Binding

• Strategies
• Tethering the chain-ends of dendrimers with
  NSB-resistant PEG moieties
• Minimizing nonspecific binding of proteins on a
  solid surface

68
Characterization of PEG-Tethered Dendrimer and
its Monolayers

• Characterization of PEG-tethered Dendrimers
• 2D-, C-, H-NMR analysis
• ? 82 conjugation ratio of PEG moiety per
  dendrimer
• FT-IR, analysis
• ? C-O-C linkage in a tethered PEG groups
• Contact-angle measurement
• Hydrophilicity

69
Non-Specific Binding Level of Proteins

• SPR measurement
• Dendrimer monolayer formation
• Protein solution (1mg/mL in PBS) treatment
• PBS washing for 10 min
• Measure the amount of protein adsorption

70
PEG-conjugated PAMAM Dendrimer Layer
Nonspecific protein adsorption test
Procedures MUA SAMs/Au Activation to give
reactive esters Covalent attachment of a
dendrimer layer Protein adsorption for 1 h
FITC-labeled BSA (1 mg/mL in PBS) FITC-labeled
human serum (1 mg/mL in PBS) Thorough washing
with PBS for a few hours Intensity measurements
with a fluorescence scanner
Human serum
BSA
29.5
11.4

• FITC-labeled human serum
• (A-1) PAMAM dendrimer monolayer
• (A-2) PEG-conjugated PAMAM dendrimer
  monolayer
• FITC-labeled BSA
• (B-1) PAMAM dendrimer monolayer
• (B-2) PEG-conjugated PAMAM dendrimer monolayer

71
Perspectives

• Promising Tool for Demonstration of Protein
  Functions
• Diagnostics
• Drug Discovery
• Understanding of Protein
  Network
• Technology Platforms for Viable Protein Chips

72
Acknowledgement
KAIST Dr. Hyun C. Yoon
Mi-Young Hong Do-Hoon Lee
Young-Ja Kim Prof.
Byung-Suk Choi POSTEC Prof. Ki-Moon Kim
Dr. Jae-Wook Lee
This work was supported by the BK21 Program of
MOE and the NRL Program of MOST, Korea.