Article Critiques Protein Microarray January 19, 2007

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<ul><li> Slide 1 </li> <li> Article Critiques Protein Microarray January 19, 2007 </li> <li> Slide 2 </li> <li> What is the role of Microarray Technologies In 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 </li> <li> Slide 3 </li> <li> Miniaturization Enables the Global Analysis of Many Molecules DNA Microarrays (gene expression) Tissue Arrays Gene expression Surface markers diagnostics Protein/Antibody Arrays (protein quantification Protein/protein interaction Protein activity/function Also: Small molecule arrays? Cell arrays? . $$$ Billion Dollar Industry $$$ </li> <li> Slide 4 </li> <li> 1 mm DNA Microarrays are now big business Single arrays are available f or all genes in Human genome 6.5 Million Probes per Array! 5 micron features </li> <li> Slide 5 </li> <li> 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 </li> <li> Slide 6 </li> <li> 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) </li> <li> Slide 7 </li> <li> Department of Zoology, University of Oxford, South Parks Road, Oxford OX1 3PS, UK. *To whom correspondence should be addressed. Email: david.rogers@zoology.ox.ac.uk 925 citations since 2000 </li> <li> Slide 8 </li> <li> EXPERIMENTAL &amp; 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 </li> <li> Slide 9 </li> <li> EXPERIMENTAL &amp; THEORETICAL METHODS (3) Fluorescent Detection Excite a molecule with light at 1 Energy lost in vibrational modes Light emitted at 2 &gt; 1 Using different fluorophores can simultaneously detect many colors Extremely sensitive (with work you can see 1 molecule!) </li> <li> Slide 10 </li> <li> Attachment Chemistries Substrate Chemical Activation (Aldehyde/NHS) Spotting Proteins Blocking Step (BSA / Glycein) </li> <li> Slide 11 </li> <li> FIGURE 1: Demonstration of Protein Spotting and Immobilization Bodiby FL-IgG Cy3 IB 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 </li> <li> Slide 12 </li> <li> 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 </li> <li> Slide 13 </li> <li> EXPERIMENTAL &amp; 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 </li> <li> Slide 14 </li> <li> 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? </li> <li> Slide 15 </li> <li> Alexa 488 BSA-DIG Cy5 BSA-Biotin Cy3 BSA-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 </li> <li> Slide 16 </li> <li> Critique Summary To be improvedGood 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) Major points Minor points </li> <li> Slide 17 </li> <li> 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 </li> <li> Slide 18 </li> <li> 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 &amp; THEORETICAL METHODS (1) Cell-Free Protein Synthesis Expression vector Polymerase Ribosomes Nucleic acids Amino acids Cofactors ATP </li> <li> Slide 19 </li> <li> EXPERIMENTAL &amp; 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 </li> <li> Slide 20 </li> <li> EXPERIMENTAL &amp; THEORETICAL METHODS (3) Biotin and Avidin (Biotech Velcro) 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! Streptavidin: tetrameric protein </li> <li> Slide 21 </li> <li> 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 </li> <li> Slide 22 </li> <li> 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 &gt; mRNA -&gt; 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) </li> <li> Slide 23 </li> <li> 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 -&gt; approximately 10 9 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 </li> <li> Slide 24 </li> <li> 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 possible 841 Only 47 interactions previously Known 17 of 20 gold standard 19 of 36 co-IP (intermediates?) </li> <li> Slide 25 </li> <li> Authors List Several Technical Challenges: 1.Bridging proteins make simple two-body interactions incomplete 2.Use of peptide tags can cause interference with binding 3.Post-translational modifications may not be captured 4.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 </li> <li> Slide 26 </li> <li> Critique Summary To be improvedGood 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 Dont 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 Major points Minor points </li> </ul>