implementation of radiotracers use in methods for differential analysis of protein expression mauro...
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Implementation of radiotracers use in methods for differential analysis of protein
expression
Mauro FasanoCentre of NeuroScience
and DBSFUniversity of Insubria
at Busto [email protected]
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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 40000 Genes 1 Million Proteins 1 Million Proteins
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40000 Genes 40000 Genes 1 Million 1 Million ProteinsProteins
• 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)
![Page 9: Implementation of radiotracers use in methods for differential analysis of protein expression Mauro Fasano Centre of NeuroScience and DBSF University of](https://reader036.vdocuments.us/reader036/viewer/2022062720/56649f0d5503460f94c21022/html5/thumbnails/9.jpg)
The classical approach
Sample solubilization
IEF
SDS - PAGE
Display of results
Gel analysis
Mass spectrometry (MALDI)
![Page 10: Implementation of radiotracers use in methods for differential analysis of protein expression Mauro Fasano Centre of NeuroScience and DBSF University of](https://reader036.vdocuments.us/reader036/viewer/2022062720/56649f0d5503460f94c21022/html5/thumbnails/10.jpg)
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)
![Page 12: Implementation of radiotracers use in methods for differential analysis of protein expression Mauro Fasano Centre of NeuroScience and DBSF University of](https://reader036.vdocuments.us/reader036/viewer/2022062720/56649f0d5503460f94c21022/html5/thumbnails/12.jpg)
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)
EE
Reagent
Excited product
LIGHTLIGHT
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Database query with peptide masses
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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
1. Differential Gel Electrophoresis
2. 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:Task 1: real-time acquisition of western blots with radiolabelled probes
• Task 2:Task 2: development of an antibody array to perform simultaneous multiple Western blots
• Task 3:Task 3: differential in-gel electrophoresis by using tracers at different energy (32P and 33P)
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Real-time western blots
B
S*S*
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Real-time western blots
Mic
rostrip
sen
sor
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Miniaturized multi-western array
B
S*S*
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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
+
+
P P 32P
P P 33P
32P
32P
32P
32P
33P
33P
Healthy
Diseased
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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)