PROTEIN ARRAYS prepared mostly by Ailie

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PROTEIN ARRAYS(prepared mostly by Ailie)
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The goals of proteomics

• Proteomics aims to simultaneously characterize
  all the proteins in biological samples
• Identify / sequence the proteins and determine
  their relative abundances
• Characterize their posttranslational
  modifications
• Determine the three dimensional structure
• Identify the interactions between the proteins,
  and with other molecules most proteins function
  in complexes
• Follow all the above during development, in
  response to hormonal stimuli, in health and
  disease, etc.

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Protein arrays can do the job

• So far, we talked about using chromatography,
  2D-electrophoresis and tandem mass spectrometry
  to achieve the goal of proteomics
• However, protein arrays are possible solution to
  the problem and might be able to provide the data
  in a fraction of the cost and a fraction of the
  time.
• Why not now?

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What is an array?

• An array is a high-density arrangement of
  capture agents located at defined positions on a
  slide.
• Arrays are to detect or measure the presence
  and/or quantity of genes or proteins in a sample.
  
• Perform a large number of tests simultaneously

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Topics for protein arrays

• Uses
• Types
• Binding
• Fabrication and printing
• Hurdles
• Exotic arrays
• Future directions

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Emili and Cagney 2000 Nature
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DNA Microarray Hybridization
Targets hybridize to complementary probes by base
pairing
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A Good Array

• Reproducible
• High density (many spots per slide)
• Maximal binding efficiency
• Homogenous signal
• Minimal background noise

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Analytical vs. Functional Protein arrays

• (a) Analytical
• Identify specific proteins/ expression of
  proteins in a complex mixture
• (b) Functional
• Identify biochemical activity
• such as specific protein-
• drug, protein-enzyme or
• protein-protein interactions.

http//www.dojindo.co.jp/letterj/106/reviews_02_su
b-01.html
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Even more complex protein arrays
http//www.stat.purdue.edu/research/coalesce/bioin
formatics/Center_for_Bioinformatics/protein_array_
analysis.html
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Reverse Array

• Cellular extracts are spotted directly onto the
  array surface to analyze entire cell at one time
• Labeled antibodies used to probe the array for
  analytes of interest in the micro-spots

• Disadvantage
• Rare proteins may not be detected
• Increased chance of unspecific reactions

http//www.zeptosens.com/ZeptoMARK/ZeptoMARK/Zepto
MARK_products.html
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Membrane Protein Arrays

• Membrane proteins are an important drug target so
  high throughput ligand affinity screening
  desirable.
• Need to print protein AND associated lipids on
  the array

Fang Y., Frutos A., and Lahiri J. 2002 Membrane
Protein Microarrays. J. Am. Chem. Soc., 124 (11),
2394 -2395
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Scope of Protein Array Applications
http//www.bioscience.org/2003/v8/d/1017/figures.h
tm
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Antibody array
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Antigen array
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Immobilizing Capture Agents

• Protein functionality must be preserved
• Binding site must remain accessible
• Proteins with different affinities/ properties
  need to be printed onto a single slide
• Provide reproducible results

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Attachment to Slide

• Glass slide treated with an aldehyde or epoxy
  reagent, which reacts with primary amines
• Adsorption to charged or hydrophobic surfaces
• Streptavidin arrayed on slide binds to
  biotinylated molecules
• His tagged proteins arrayed on nickel coated
  slides

http//arrayit.com/Products/Substrates
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Immobilizing Capture Agents

• Covalent interaction with the surface
• Definite arranged order of molecules
• Possibility of spacers to prevent specific
  interactions
• Strong interaction between surface and capture
  agent
• Non covalent interaction with the surface
• Hydrophobic, H-bond, van der Waals,
  electrostatic, physical adsorption
• No modification to capture molecule required
• Weaker interaction between surface and capture
  agent

http//www.ciphergen.com/MasterPage.aspx?URL/tech
apps/pc/tech/arrays
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Slide Surface

• 1-Dimensional
• Capture agent binds by electrostatic/covalent
  interaction
• 2-Dimensional
• Capture agent attached to slide adhering
  molecule (e.g. Self-assembled monolayer -
  spontaneous adsorption of alkanthiols on gold
  surfaces )
• 3-Dimensional
• (3D) gel or membrane-coated surfaces such as
  polyacrylamide, agarose and nitrocellulose

Kusnezow, W et al 2003 J. Mol. Recognit. 16
165176
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Hydrophobic surface (Zeta Grip)
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Gold array
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Printing - Technicalities

• Protein drop cannot dry out (add glycerol)
• Hydrophilic spots on hydrophobic surfaces
  prevents mixing of aqueous spots
• Resolution how many drops per unit space
• Filter arrays drops disperse taking more space
• 3D arrays more slide surface area gives more
  capacity

http//www.genome.org/content/vol7/issue10
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Probe Printing - Contact

• Probes spotted onto the slide by lowering the
  printing tip to just touch the surface of the
  slide.

http//bric.postech.ac.kr/bbs/biostat/2001/2001070
3_2.html
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Probe Printing - Piezoelectric effect

• Non contact printing
• based on ink-jet printing technology
• Piezoelectric effect
• Electric current ? Material expands? Drop pushed
  out

http//teched.vt.edu
http//bric.postech.ac.kr/bbs/biostat/2001/2001070
3_2.html
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Photolithography (AFFYMEX)

• Photosensitive protection
• Mask
• UV light removes the protecting groups in
  unmasked areas
• Nucleotide added to de-protected areas.
• Repeat process

http//www.affymetrix.com
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Developing TechnologyArray Creation by
electrospray

• IDEA Deposition of proteins onto array surfaces
  after mass spectrometry analysis of sample
• METHOD Proteins separated and spotted according
  to mass using a soft landing technique after MS
  identification.
• RESULTS
• A mixture of cytochrome c, lysozyme, insulin and
  apomyoglobin arrayed by this technique and
  function confirmed by specific substrate binding
• PROBLEMS
• Time consuming to deposit enough protein.
• Loss of activity due to harshness of MS still a
  problem

• AIM Dramatically increase the complexity of the
  protein mixture applied, deposited and analyzed

Washburn, M. 2003. Soft Landing for Protein
Chips. Nature Biotechnology. 211156-57
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Protein Array Capture Agents
http//neurolab.fr/Proteo3.gif

• ANTIBODIES - natural binders of proteins
• APTAMERS - oligonucleotides with affinity for
  individual protein molecules
• - easy to synthesis, stable, robust
• MOLECULES - small molecules are easily
  synthesized and robust
• PROTEINS - important in functional analysis
• - easily denatured
• - hard to isolate large libraries
• ENZYMES, DNA, MEMBRANE PROTEINS, TOTAL CELLS..

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Antibodies as Capture Agents

• Well established ELISA technology applicable in
  array format
• Monoclonal, polyclonal or recombinant antibodies
  (scFv)
• Polyclonal antibodies recognize different
  epitopes reducing selectivity
• Single Chain Antibodies (scFv)
• The variable regions of the heavy and light
  chains can be fused together with antigen
  specificity maintained
• Smaller, easier to handle, cheaper

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Synthesized Molecules as Capture Agents

• Aptamers and Small Molecules
• Easily synthesized on large scales
• Stable and robust
• non-specific protein stains can be used for
  detection (since targets are the only proteins on
  array)
• Peptide Aptamers
• Thioredoxin small stable
• protein with variable surface
• loop that can be engineered
• as binding site to various
• targets

http//www.docforum.tm.fr/documents/25janv05propin
dusvalsavInterB.Rudkin.pdf
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Incubation of Sample and Array

• Plate conditions need to be homogeneous
• Proteins are sensitive to ambient conditions
• hard to array many different proteins and keep
  all functional.
• Biological samples are dynamic
• Cellular conditions continually changing affect
  interactions. This is hard to simulate in vivo

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DNA Microarray Signal Detection
http//www.awi-bremerhaven.de/Biomeer/molecular-ge
netics-top08-e.html

• Fluorescence or luminescence detection
• The relative intensity of label signals
  correlates to relative abundance of corresponding
  gene.

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Protein Microarray
Complex protein mixture
Capture Antibodies
Chip surface
Feature I
Feature II
1. Wash array 2. add detection
antibody
3. wash
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Detection of Protein Target Binding

• CONSIDERATIONS
• Accuracy, Efficiency, Sensitivity, Maintain
  integrity of sample
• METHODS
• Direct labels - fluorescent dyes
• Indirect labels - secondary antibodies (sandwich
  assay/ELISA)
• MALDI-TOF mass spectrometry
• Surface plasma resonance
• Rolling circle PCR
• Atomic Force Microscopy (AFM)

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Strepavidin-biotin Array based ELISA (Mendoza99)
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Detection Methods
www.functionalgenomics.org.uk/ sections/resources/
Protein_arrays.htm
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Mass Spectrometry

• Idea Detect protein bound to array without need
  for labeling
• Allows analysis of detected protein
• Method
• Add energy absorbing molecules (MATRIX) to array
  spot
• Analysis of spot by standard MALDI-TOF
• Problems
• Low throughput

http//www.evms.edu/vpc/seldi/seldiprocess/
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Surface Plasma Resonance

• The refractive index at the surface of a gold
  coated chip is monitored.
• Binding to the array probes changes this
  refractive index and allows not only for
  detection of binding but for monitoring of
  association/dissociation rates
• Advantage no labeling necessary within the array

http//www.uni-ulm.de/aktuelles/aktuelles_thema/ak
tuell0207/cast/spr.jpg
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Rolling Circle PCR

• Aim
• To improve sensitivity of detection
• Idea
• Reporter antibodies are covalently attached to
  unique oligonucleotide primers.
• A DNA circle is hybridized for a rolling circle
  amplification of the tag.
• The tag is labeled by hybridization with
  fluorescently-labeled oligonucleotides (or
  alternatively the fluorescently-labeled
  oligonucleotides can be used in the
  amplification)

Schweitze, B et al. Proc Natl Acad Sci USA
9710113-10119.
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Luminescence detection Kodadek 01
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Protein chips (MacBeath2000)
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Large scale arrays of proteins (MacBeath2000)
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Protein chips with CBD
Enzymatic one color development
Enzymatic two color development
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Transfected cell arrayBailey, Wu and Sabatini
DDT 02
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Membrane protein array Beaumont and Negulescu 99
Nature
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Bouwman..Hanash, Serological Array, Proteomics
03
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ScFv antibody array, De Wildt, Nature 2000
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The effect of multi protein complexes Kodadek 01
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FACS as array (Nolan 2008)
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Future directions

• Proteomics in a drop of blood, saliva, urine,
  sweat, pond, environmental analysis
• Bedside and clinic assay
• Rapid answers
• Screen the entire population for all their future
  diseases
• Remote medicine
• Food analysis
• Single cell assays
• More idea?