DAH3.4 Protein interactions, sub –cellular locations and arrays

1. DAH3.4 Protein interactions, sub –cellular locations and arrays Kathryn Lilley Cambridge Centre for Proteomics Department of Biochemistry University of Cambridge k.s.lilley@bioc.cam.ac.uk www.bio.cam.ac.uk/proteomics/ Part III Systems Biology

2. Outline • Protein-protein interactions • Protein sub-cellular location • Protein Arrays • Limitations of proteomics and systems biology

3. Types of protein analysis Abundance Isoform status Quantitative Mass Spec Western blotting GFP tagging Immuno- histochemistry Enzyme assay Arrays Mass Spectrometry Western blotting Functional arrays Proteins present PPI SCL Function Mass Spectrometry Western blotting GFP tagging Immuno- histochemistry Enzyme assay Arrays Y2H Tagging + Mass Spectrometry Western blotting Structural studies Protein Arrays Biophysical assays (e.g.ITC, AUC) Mass Spectrometry GFP tagging Immuno- histochemistry Enzyme assay Functional arrays Enzyme assay Genetic approaches

4. Protein-Protein Interactions PPI Identification of protein binding partners Y2H Tagging + Mass Spectrometry Western blotting Structural studies Protein Arrays Biophysical assays (e.g.ITC, AUC)

5. Methods to determine PPI Y2H (yeast two hybrid) Tagging + Mass Spectrometry Western blotting Structural studies Protein Arrays Biophysical assays (analytical ultracentrifugation)

6. Y2H AD Gal4 transcription factor has two domains BD Lac z protein (can easily assayed using x-gal) mRNA AD BD Lac Z gene UAS If BD and AD are expressed separately, no transcription If BD and AD are expressed as fusion proteins with proteins that interact that are brought close together structurally and can bring about transcription of LacZ AD Bait Prey BD

7. Two-hybrid screen for protein partners Bait and prey libraries expressed in yeast Bait and prey can come from any organisms but must be fused to yeast AD and BD Only any good for binary interactions Very high false discovery rate

8. Protein Complex Purification • Methods mostly based around fusion of bait protein and selective purification of complex by immunoprecipitation • Methods can be ‘dirty’

9. Tag bait protein and pull it and its preys out of a mixture Tag could be avidin (binds to Biotin) hexa histidine (binds to Ni-NTA) flag (small peptide epitope for which and antibody is available) Pull-down Wash Elute Two potential problems: All single step purifications – many extraneous protein co-purified. Tagged proteins often over expressed.

10. Tandem Affinity Purification Tagging PROTEIN SPACER CBP TEV site PROTEIN A Bind and wash IgG beads PROTEIN SPACER CBP TEV site PROTEIN A Run purified complex on 1D gel Identify components by MS TEV protease Bind and wash Calmodulin beads PROTEIN SPACER CBP Elute with Calcium

11. iPAC (parallel affinity chromatography) F S Negative control with no tags Bait with two tags Flag and Strep FLAG M2 purification, FLAG peptide elution FLAG M2 purification, FLAG peptide elution Strep purification, Biotinelution Strep purification, Biotin elution Rees et al Mol.Cell Prot 2011

12. Types of protein analysis Abundance Isoform status Quantitative Mass Spec Western blotting GFP tagging Immuno- histochemistry Enzyme assay Arrays Mass Spectrometry Western blotting Functional arrays Proteins present PPI SCL Function Mass Spectrometry Western blotting GFP tagging Immuno- histochemistry Enzyme assay Arrays Y2H Tagging + Mass Spectrometry Western blotting Structural studies Protein Arrays Biophysical assays (e.g.ITC, AUC) Mass Spectrometry GFP tagging Immuno- histochemistry Enzyme assay Functional arrays Enzyme assay Genetic approaches

13. Mass Spectrometry GFP tagging Immuno- histochemistry Enzyme assay Sub-cellular location

14. GFP tagging of yeast proteome GFP tagged proteins 75% of the yeast proteome classified to 22 distinct location Huh et al, 2003

15. Systems wide immuno-histochemistry Antibodies to 488 proteins applied to 3 different human cell lines and images stored and publically accessible Blue = DAPI staining of nucleus Barbe et al, 2008

16. Proteomics approaches Gentle cell lysis Pure fraction Subtractive proteomics LOPIT Invariant rich fraction Protein correlation profiling

17. Pure Fraction Extract proteins Parts list Digest LC-MS/MS

18. Subtractive Proteomics Boisvert et al (2010) Mol. Cell Prot 9:457

19. Invariant Rich Fraction RM SM Rough microsomes Smooth microsomes Golgi Apparatus ‘enriched fractions’ RM + SM Golgi Gilchrist et al (2006) Cell 127:1265

20. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 17 18 19 20 Distribution Profiling Gtl-6 Golgi Cyt b5 sER Sec12 ER Carbonate wash Tom Dunkley

21. LC-MS/MS (SCX/RP) Localisation of organelle proteins using isotope tagging (LOPIT) LOPIT Protocol Quantification Identification Dunkley et al (2006) Proc. Natl. Acad. Sciences 103:6518

22. PCA Analysis of iTRAQ Dataset Marker Predicted marker LOPIT assigned no location assigned +

23. Types of protein analysis Abundance Isoform status Quantitative Mass Spec Western blotting GFP tagging Immuno- histochemistry Enzyme assay Arrays Mass Spectrometry Western blotting Functional arrays Proteins present PPI SCL Function Mass Spectrometry Western blotting GFP tagging Immuno- histochemistry Enzyme assay Arrays Y2H Tagging + Mass Spectrometry Western blotting Structural studies Protein Arrays Biophysical assays (e.g.ITC, AUC) Mass Spectrometry GFP tagging Immuno- histochemistry Enzyme assay Functional arrays Enzyme assay Genetic approaches

24. Function • Functional genomics • One-by one knock out and observe phenotype • Kamath et al, 2003, used RNAi to inhibit the function of, 86% of the 19,427 predicted genes of C. elegans and identified 1,722 mutant phenotypes • Karlas et al 2010, screened a genome-wide siRNA library consisting of approximately 62,000 siRNAs targeting ,17,000 annotated genes and ,6,000 predicted genes in conjunction with infection of a lung epithelial cell line with H1N1 virus. They found that 287 host cell genes influence infection. • In silico predictions • Sequence alignment • Structure alignment • Functional Arrays • e.g. Kinase substrates.calmodulin binding, …… Functional arrays Enzyme assay Genetic approaches

25. Protein Arrays Three types: Functional Arrays Analytical/Diagnostic Arrays Tissue Arrays

27. How to make them Slides derivatised with the following on to which proteins can be spotting using contact printing methods Nitrocellulose Amino silane Gold coated Bifunctional cross linkers Biotin/avidin hexa-histidine/ Nickel (Ni-NTA)

28. Synthesis of a yeast protein array Proteins expressed with a tag which makes them easy to purify Still a major task to express and purify every proteins from an organism Mok et al, 2009

29. Protein in situ arrays (PISA)

30. Nucleic Acid Programmable Protein Array (NAPPA)

31. DNA Array to Protein Array (DAPA) Protein DNA

32. New approach? He et al (2009) New Biotechnology 26:227

33. Detection Fluorescence Radioactivity Quantum dots Surface plasmon resonance Mass spectrometry

34. Functional arrays

35. Kinase Substrates For more examples see Chen and Snyder (2010) J. Proteomics 74:2147-57

36. Analytical/Diagnostic Arrays Protein Expression Profiling on Capture Arrays

37. Antibody Affinity Arrays A2M2S Affinity Arrays and MALDI Mass Spectrometry Darii et al, 2009

38. Tissue Arrays • Tissues mounted to standard silanized slides. • Formalin-fixed paraffin-embedded human tissues • Used for immunohistochemistry, in situ hybridization, fluorescent in situ hybridisation and in situ PCR • Available from different companies supplying cancerous and normal tissues

39. Human Protein Atlas www.proteinatlas.org Tissue arrays, 48 normal tissue types, 20 cancers. Cell line arrays – 47 different cell lines Immunohistochemitsry - Sections of tissues Immunohistochemistry of 3 cell lines Antibodies raised against expressed sequence tags , a specific epitope for each human protein PrESTs

40. Strengths Under represented proteins present Functional information High throughput Weaknesses Proteins need to be in correct orientation and in vivo relevant structure Unknown splicing variants not detected Mixed PTMs not detected Co-interacting partners not detected Off target interactions High cost of manufacture

41. Problems with proteomics and systems biology Tip of ice berg Combinatorial PTMs Keeping complexes together Tissue specificity Synchronisation of cells Singel cell proteomics Whole proteome is highly dynamic Often not enough samples/resources to carry out sufficient replicate experiments

42. Single Cell Proteomics ICPMS Inductively coupled plasma MS Bendall et al (2011) Science