Transcript Slide 1

Article Critiques
Protein Microarray
January 19, 2007
What is the role of Microarray Technologies
In Large-Scale Biology?
Global Monitoring of Information Flow
In Biology
DNA: (3 billion bases)
mRNA: (30,000 – 100,000 genes
+ ???? Non-coding)
Proteins: (100,000 - 300,000)
Questions:
What are the genes in the cell?
How are these regulated?
What are the proteins?
What do they do?
How to they fit together….
Miniaturization Enables the Global
Analysis of Many Molecules
$$$
Billion Dollar Industry
$$$
Tissue Arrays
Gene expression
Surface markers
diagnostics
DNA Microarrays
(gene expression)
Also:
Small molecule arrays?
Cell arrays?
….
Protein/Antibody Arrays
(protein quantification
Protein/protein interaction
Protein activity/function
DNA Microarrays are now
big business
1 mm
Single arrays are available f
or all genes in
Human genome
6.5 Million Probes per Array!
5 micron features
Typical Flow for DNA Microarray
(Relative Gene Expression)
Start with two samples
For relative comparison
Break open cells and
Isolate mRNA
Label Cells with different
Color Fluorescent Molecules
Hybridize to Array and
wash
Protein Microarrays
• Tool for determining protein function/interaction
• Some commercial products recently available but still a cottage industry
Why: Proteins are much more challenging for micro array applications
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Expression (how do you make 30,000 proteins?)
Purification
Proteins have very different physical properties
Proteins are dynamic : post translational modifications, complexes…
Many interesting proteins are not soluble
Stability (must keep hydrated, storage can be problem)
Attachment to array ( correct orientation, folding)
Department of Zoology, University of Oxford, South Parks Road,
Oxford OX1 3PS, UK.
*To whom correspondence should be addressed. Email:
[email protected]
925 citations since 2000
EXPERIMENTAL & THEORETICAL METHODS (1)
Microarray Spotting
• Robot used to generate arrays of
molecules
• Molecules are typically arrayed by
“contact dispensing” - dipping pins into
well and touching on slides
• Pins are specially designed to allow
for high spatial precision in spotting
(typically ~ 100 microns
• Slide is “functionalized” to allow for
attachment of molecules to spots
EXPERIMENTAL & THEORETICAL METHODS (3)
Fluorescent Detection
• Excite
a molecule with light at λ1
• Energy lost in vibrational modes
• Light emitted at λ2 > λ1
• Using different fluorophores can
simultaneously detect many
colors
• Extremely sensitive (with work
you can see 1 molecule!)
Attachment Chemistries
Substrate
Chemical
Activation
(Aldehyde/NHS)
Spotting
Proteins
Blocking Step
(BSA / Glycein)
Application 1: Protein / Protein Interactions
FIGURE 1: Demonstration of Protein Spotting and Immobilization
Bodiby FL-IgG
• Use of 40% glycerol to avoid drying
Cy3 – IκBα
• Simultaneous and specific detection
of proteins using three-color
fluorescence – proteins are folded
Cy5 – FKB12
+ rapamycin
Protein P50 FRB
G
• 10,000 spots per slide
Cy5 – FKB12
- rapamycin
• Authors discuss concentration for
spotting but not the volume of drops!
A+B+C
• Proteins pairs chosen are very
stable and have high affinities (not a
realistic example!)
FIGURE 2: Demonstration of Scalability of Array technique
“works across the whole slide”
Otherwise nothing new hear over figure 1.
Single FRB in
Background of
Protein G
EXPERIMENTAL & THEORETICAL METHODS (4)
Looking for Kinase Targets…
Target Protein
Protein KINASE
Phosphorylated (activated)
Target Protein
Area of great interest in pharmaceutical sciences : Inhibiting
Kinases: Example Gleevec
FIGURE 3: Detection of Kinase Substrates
• Use of BSA-NHS slides (chemistry 2)
PKA
CKII
p42
• Radioactive phosphate used in
reaction (ISOTOPIC LABELING)
• Interesting method of detection by
dipping in photographic emulsion
• Again – these are very well-known and
highly active protein substrates
• why are spots so different in
size/uniformity?
Alexa 488
BSA-DIG
• Small molecules are attached to BSA
– major limitation
Cy5
BSA-Biotin
• Significant cross-talk in C is not
addressed
Cy3
BSA-AP1497
ALL Above
• Small molecules are once again
chosen to be very easy
• Potentially useful way to screen for
specificity on candidate drugs
Critique Summary
To be improved
Minor points
Major points
Examples are all too easy
Not enough proteins used – will it work
over a huge panel of proteins?
Do not address detection limit in terms
of weaker interaction
Good
Good proof of concept
demonstration – various
applications
Attachment chemistry and
detection methods are key and
are well described
Do not address some technical points
such as spot contamination, cross-talk,
array storage…
Put in context of current s-o-a
Overly optimistic on generality
of this technology (see above)
Admit major issues in
generating protein
Not quantitative enough
regarding concentrations.
Prophesize the use of cell-free
synthesis (next paper)
1Harvard Institute of Proteomics, Department of Biological
Chemistry and Molecular Pharmacology, Harvard
Medical School, 320 Charles Street, Cambridge,
MA 02141, USA.
68 citations since 2006
EXPERIMENTAL & THEORETICAL METHODS (1)
Cell-Free Protein Synthesis
Clone Gene
Insert into Expression Vector
Insert Vector into Cells
Screen Cells for Vector
Culture Cells
Expression vector
Polymerase
Ribosomes
Nucleic acids
Amino acids
Cofactors
ATP…
Sequence Gene
Express protein?
Purify Protein
Spot on Array
$$$$
+
TIME
EXPERIMENTAL & THEORETICAL METHODS (2)
Protein Detection (Epitope Tags)
Versitile strategy for detecting and purifying
proteins expressed by cloned genes.
Gene
Proteins are genetically engineered with an
additional peptide, creating a fusion protein
Tag
Protein Expression
Take advantage of well-developed
antibodies that are highly specific to the
expressed tag
Eliminate need to produce antibodies (big
pain in the butt)
Common tags include: glutathione-Stransferase (GST), c-myc, 6-histidine (6XHis), FLAG®, green fluorescent protein
(GFP), maltose binding protein (MBP),
influenza A virus haemagglutinin (HA), bgalactosidase, and GAL4.
Protein
Tag
EXPERIMENTAL & THEORETICAL METHODS (3)
Biotin and Avidin (Biotech Velcro)
Streptavidin: tetrameric protein
Biotin: Vitamin H or B7
Small Molecule
Many techniques for adding this
to proteins or DNA - using enzymes
or UV light, or chemical techniques
one of the strongest biological and noncovalent interactions known
Kd ~ 10-14 M/L
Used everywhere in biotechnology!
Main Point of Paper
• Address or Eliminate problems in protein
expression/purification/storage by in situ
synthesis
• Massive reduction in expense of producing
proteins
• Genetically engineered epitope tags used
for an automatic purification and allow for
immobilization and quantificaiton of
proteins
FIGURE 1: Scheme for in situ synthesis of proteins on array
Spot cDNA expression
Vector onto array
With Polyclonal anti-GST
Cell-Free Protein
Expression
cDNA –> mRNA -> Protein
Detect and Quantify Protein
With monoclonal GST
Points:
a. No storage of proteins for array (can store dry!)
b. “Easy” to generate expression vectors
c. 900 micron spacing (500/slide)
Questions:
a. How much of protein is captured (diffusion carries some away?)
b. Similarly, there is a fundamental limit on feature density.
c. What happens to Avidin or anti-GST once slide is dried?
FIGURE 2: Protein Array Generation and Interactions
MAB GST
JUN
p16
• Gene expression efficiency varied approximately 25%
• Suggested that cDNA concentration could be used to adjust this (scaleable?)
• 10 fM per spot -> approximately 109 molecules
• NOTE: B and C are difficult to make out but there appears to be background spots
• Large error bars (Standard Deviation) for p16 queries
FIGURE 3: Biological Application (replication complex)
• 29 genes used for array
• Expression ranged over 10x
(worse than in previous test)
• Looked at all 29 possible twoprotein interactions (2x repeats)
on 29 array slides.
• Found 110 interactions of possible
841
• Only 47 interactions previously
Known
• 17 of 20 “gold standard”
• 19 of 36 co-IP (intermediates?)
Authors List Several Technical Challenges:
1.
2.
3.
4.
Bridging proteins make simple two-body interactions incomplete
Use of peptide tags can cause interference with binding
Post-translational modifications may not be captured
Lack of spatial compartmentalization (some proteins never see eachother!)
Additional Technical Challenges:
1. Unlikely that a single condition/cell extract will allow efficient translation of
a large number of proteins.
2. Array density is rather low (due to diffusion during synthesis?)
3. What are limits in terms of binding affinity?
4. Non-specific or biologically irrelevant interactions are difficult to determine
Other notes:
1. Would seem that you could use different query proteins or combinations
of proteins in different areas – look at multi-body interactions
Critique Summary
To be improved
Major points
Description of attachment chemistry is
limited and drying of antibodies seems
strange (but seems to work)
Again: Not enough proteins used – will
it work over a huge panel of proteins?
Very clever idea that addresses
a major problem in protein
arrays
Ability to extend beyond simple
two-body interactions
Again: Do not address detection limit in
terms of weaker interaction
Don’t address some large spot-spot
variabilities
Minor points
Good
Some figures are difficult to
make out ( figure 2b, 2c)
Good demonstration of
biological application
Admit some limitations
Well referenced
Figure 1 could should more
steps