1. 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@uninsubria.it

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

3. 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

4. Post-translational modifications Splice variants Proteolytical processing

5. Proteome is dynamic…

6. The classical approach  Sample solubilization  IEF  SDS - PAGE  Display of results  Gel analysis  Mass spectrometry (MALDI)

7. The classical approach  Sample solubilization  IEF  SDS - PAGE  Display of results  Gel analysis  Mass spectrometry (MALDI) UREA 7M, THIOUREA 2M, CHAPS 4%

8. The classical approach  Sample solubilization  IEF  SDS - PAGE  Display of results  Gel analysis  Mass spectrometry (MALDI)

9. The classical approach  Sample solubilization  IEF  SDS - PAGE  Display of results  Gel analysis  Mass spectrometry (MALDI)

10. The classical approach  Sample solubilization  IEF  SDS - PAGE  Display of results  Gel analysis  Mass spectrometry (MALDI) Immunoblotting Coomassie Blue or silver staining

11. The classical approach  Sample solubilization  IEF  SDS - PAGE  Display of results  Gel analysis  Mass spectrometry (MALDI)

12. The classical approach  Sample solubilization  IEF  SDS - PAGE  Display of results  Gel analysis  Mass spectrometry (MALDI)

13. Protein identification Immunoblot (against selected proteins) Digest, MS & database query Digest & MS/MS

14. Immunoblot (Western blot) E Reagent Excited product LIGHT

15. Database query with peptide masses

17. MS/MS

18. 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

19. Making it simpler… Subcellular fractionation Affinity tags Enrich for specific post-translational modifications Protein-protein interaction (Interactomics)

20. Quantitative proteomics 1.Differential Gel Electrophoresis 2.Gel-free MS with ICAT

21. DIGE ®

23. Isotope-coded affinity tagging

24. Phosphoproteomics Enrichment of phosphopeptides Immunoblot (anti-pSer, etc.) or in vitro labeling with 32 P

25. 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 ( 32 P and 33 P)

26. Real-time western blots B S*

27. Microstrip sensor

28. Miniaturized multi-western array B S*

29. Spotting antibodies on a membrane 120000 spots on a 25 x 75 mm slide (64 spots per mm 2 )

30. RadioDIGE + + PP 32 P PP 33 P   32 P 33 P Healthy Diseased

31. Mix and separate by 2D-PAGE 32 P 33 P

32. Other Matters Multiplexing protein-protein interactions –Assays in solution –Zeptosens CeLyA –Biacore affinity chip –Peptide chips

33. 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 (Ta 2 O 5 ). This high- refractive-index material guides laser light on the surface of the chip only, permitting selective detection of captured labels.

34. The Biacore Approach Biacore, Uppsala, Sweden

35. 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 32 P-ATP Jerini peptide technol., Berlin

36. 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)