Implementation of radiotracers use in methods for differential analysis of protein expression

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Implementation of radiotracers use in methods for
differential analysis of protein expression

• Mauro Fasano
• Centre of NeuroScience
• and DBSF
• University of Insubria
• at Busto Arsizio
• mauro.fasano_at_uninsubria.it

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Outline

• Background, state of the art
• (adapted from my presentation at the Varese
  meeting)
• The toy-project
• Update in protein arrays technology

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Proteome

• The set of proteins encoded by the genome
• The set of all p. isoforms, their modifications,
  their interactions
• The set of p. as above in relationship to a given
  state (disease, treatment, time, etc.)
• 40000 Genes ? 1 Million Proteins

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40000 Genes ? 1 Million Proteins

• Post-translational
• modifications
• Splice variants
• Proteolytical processing

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Proteome is dynamic
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The classical approach

• Sample solubilization
• IEF
• SDS - PAGE
• Display of results
• Gel analysis
• Mass spectrometry (MALDI)

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The classical approach

• Sample solubilization
• IEF
• SDS - PAGE
• Display of results
• Gel analysis
• Mass spectrometry (MALDI)

UREA 7M, THIOUREA 2M, CHAPS 4
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The classical approach

• Sample solubilization
• IEF
• SDS - PAGE
• Display of results
• Gel analysis
• Mass spectrometry (MALDI)

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The classical approach

• Sample solubilization
• IEF
• SDS - PAGE
• Display of results
• Gel analysis
• Mass spectrometry (MALDI)

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The classical approach

• Sample solubilization
• IEF
• SDS - PAGE
• Display of results
• Gel analysis
• Mass spectrometry (MALDI)

• Immunoblotting
• Coomassie Blue or silver staining

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The classical approach

• Sample solubilization
• IEF
• SDS - PAGE
• Display of results
• Gel analysis
• Mass spectrometry (MALDI)

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The classical approach

• Sample solubilization
• IEF
• SDS - PAGE
• Display of results
• Gel analysis
• Mass spectrometry (MALDI)

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Protein identification

• Immunoblot (against selected proteins)
• Digest, MS database query
• Digest MS/MS

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Immunoblot(Western blot)
LIGHT
Excited product
Reagent
E
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Database query with peptide masses
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(No Transcript)
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MS/MS
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Limits of the classical approach

• Too many proteins in the sample
• Only the most abundant proteins are displayed and
  identified
• Small hydrophobic proteins are not adequately
  separated
• Low-abundance proteins are hindered by more
  abundant ones
• Gels are not always reproducible

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Making it simpler

• Subcellular fractionation
• Affinity tags
• Enrich for specific post-translational
  modifications
• Protein-protein interaction (Interactomics)

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Quantitative proteomics

• Differential Gel Electrophoresis
• Gel-free MS with ICAT

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DIGE
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DIGE
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Isotope-coded affinity tagging
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Phosphoproteomics

• Enrichment of phosphopeptides
• Immunoblot (anti-pSer, etc.)

or

• in vitro labeling with 32P

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The toy project

• Task 1 real-time acquisition of western blots
  with radiolabelled probes
• Task 2 development of an antibody array to
  perform simultaneous multiple Western blots
• Task 3 differential in-gel electrophoresis by
  using tracers at different energy (32P and 33P)

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Real-time western blots
S
B
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Real-time western blots
Microstrip sensor
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Miniaturized multi-western array
S
B
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Spotting antibodies on a membrane
120000 spots on a 25 x 75 mm slide (64 spots per
mm2)
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RadioDIGE

32P
?
32P
P
P
32P
Healthy
32P
32P

33P
?
P
P
Diseased
33P
33P
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Mix and separate by 2D-PAGE
32P
33P
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Other Matters

• Multiplexing protein-protein interactions
• Assays in solution
• Zeptosens CeLyA
• Biacore affinity chip
• Peptide chips

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Cell Lysate Assay
Witterswil, Switzerland-based Zeptosens has
developed a method based on planar waveguides
(PWGs), modifying the standard glass-slide
substrate with a thin film of tantalum pentoxide
(Ta2O5). This high-refractive-index material
guides laser light on the surface of the chip
only, permitting selective detection of captured
labels.
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The Biacore Approach
Biacore, Uppsala, Sweden
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The Jerini approach

• Take the enzyme (e.g., kinase)
• Scan databases for potential targets
• Synthesize and array 20000 peptides on a chip
• Incubate with the kinase and 32P-ATP

Jerini peptide technol., Berlin
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Further readings

• Nature Insight in the 13 March 2003 issue (Vol.
  422, p. 191 and ff.)
• Proteomics in multiplex. Nature 429, 101-107
  (2004)
• Protein Microarrays Mature. The Scientist 18, 42
  (August 2004 issue)