Article CritiquesProtein Microarray
January 19, 2007
What is the role of Microarray TechnologiesIn Large-Scale 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….
Global Monitoring of Information Flow
In Biology
Miniaturization Enables the Global Analysis of Many Molecules
DNA Microarrays(gene expression)
Tissue ArraysGene expressionSurface markersdiagnostics
Protein/Antibody Arrays(protein quantificationProtein/protein interactionProtein activity/function
Also:Small molecule arrays?Cell arrays?….
$$$Billion Dollar Industry
$$$
1 mm
DNA Microarrays are now big business
Single arrays are available for 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 samplesFor relative comparison
Break open cells and Isolate mRNA
Label Cells with different Color Fluorescent Molecules
Hybridize to Array andwash
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
• 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)
SpottingProteins
Blocking Step(BSA / Glycein)
FIGURE 1: Demonstration of Protein Spotting and Immobilization
Bodiby FL-IgG
Cy3 – IκBα
Cy5 – FKB12+ rapamycin
Cy5 – FKB12- rapamycin
A + B + C
Protein P50 FRB G
• Use of 40% glycerol to avoid drying
• Simultaneous and specific detection of proteins using three-color fluorescence – proteins are folded
• 10,000 spots per slide
• Authors discuss concentration for spotting but not the volume of drops!
• Proteins pairs chosen are very stable and have high affinities (not a realistic example!)
Application 1: Protein / Protein Interactions
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
Phosphorylated (activated)Target Protein
Protein KINASE
Area of great interest in pharmaceutical sciences : Inhibiting Kinases: Example Gleevec
FIGURE 3: Detection of Kinase Substrates
PKA
CKII
p42
• Use of BSA-NHS slides (chemistry 2)
• 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 488BSA-DIG
Cy5BSA-Biotin
Cy3BSA-AP1497
ALL Above
• Small molecules are attached to BSA – major limitation
• Significant cross-talk in C is not addressed
• Small molecules are once again chosen to be very easy
• Potentially useful way to screen for specificity on candidate drugs
Critique SummaryTo be improved Good
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
Do not address some technical points such as spot contamination, cross-talk, array storage…
Good proof of concept demonstration – various applications
Attachment chemistry and detection methods are key and are well described
Put in context of current s-o-a
Overly optimistic on generality of this technology (see above)
Not quantitative enough regarding concentrations.
Admit major issues in generating protein
Prophesize the use of cell-free synthesis (next paper)
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1Harvard Institute of Proteomics, Department of BiologicalChemistry and Molecular Pharmacology, HarvardMedical School, 320 Charles Street, Cambridge,
MA 02141, USA.68 citations since 2006
Clone Gene
Insert Vector into Cells
Screen Cells for Vector
Culture Cells
Spot on Array
Insert into Expression Vector
Sequence Gene
Express protein?
Purify Protein
$$$$ +TIME
EXPERIMENTAL & THEORETICAL METHODS (1)
Cell-Free Protein Synthesis
Expression vectorPolymeraseRibosomesNucleic acidsAmino acidsCofactorsATP…
EXPERIMENTAL & THEORETICAL METHODS (2)
Protein Detection (Epitope Tags)
Versitile strategy for detecting and purifying proteins expressed by cloned genes.
Proteins are genetically engineered with an additional peptide, creating a fusion protein
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-S-transferase (GST), c-myc, 6-histidine (6X-His), FLAG®, green fluorescent protein (GFP), maltose binding protein (MBP), influenza A virus haemagglutinin (HA), b-galactosidase, and GAL4.
Gene Tag
Protein Expression
Protein Tag
EXPERIMENTAL & THEORETICAL METHODS (3)
Biotin and Avidin (Biotech Velcro)
Biotin: Vitamin H or B7Small MoleculeMany techniques for adding this to proteins or DNA - using enzymesor UV light, or chemical techniques
one of the strongest biological and noncovalent interactions knownKd ~ 10-14 M/L
Used everywhere in biotechnology!
Streptavidin: tetrameric protein
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 arrayWith Polyclonal anti-GST
Cell-Free Protein ExpressioncDNA –> mRNA -> Protein
Detect and Quantify Protein With monoclonal GST
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?
Points: a. No storage of proteins for array (can store dry!) b. “Easy” to generate expression vectors
c. 900 micron spacing (500/slide)
FIGURE 2: Protein Array Generation and Interactions
• 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
MAB GST JUN p16
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 two-protein interactions (2x repeats)on 29 array slides.
• Found 110 interactions of possible841
• Only 47 interactions previously Known
• 17 of 20 “gold standard”
• 19 of 36 co-IP (intermediates?)
Authors List Several Technical Challenges:
1. Bridging proteins make simple two-body interactions incomplete2. Use of peptide tags can cause interference with binding3. Post-translational modifications may not be captured4. 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 combinationsof proteins in different areas – look at multi-body interactions
Critique SummaryTo be improved Good
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?
Again: Do not address detection limit in terms of weaker interaction
Don’t address some large spot-spot variabilities
Very clever idea that addresses a major problem in protein arrays
Ability to extend beyond simple two-body interactions
Good demonstration of biological application
Some figures are difficult to make out ( figure 2b, 2c)
Figure 1 could should more steps
Admit some limitations
Well referenced
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