1. Proteomics Informatics – Protein Characterization II: Protein Interactions (Week 11)

2. Discovering New Protein Interactions with Affinity Capture Mass Spectrometry A B A C D Digestion Mass spectrometry E F Identification

3. More / better quality interactions Affinity Capture Optimization Screen + Cell extraction Lysate clearance/ Batch Binding Binding/Washing/Eluting SDS-PAGE Filtration

4. Analysis of Non-Covalent Protein Complexes Taverner et al., Acc Chem Res 2008

5. Non-Covalent Protein Complexes Schreiber et al., Nature 2011

6. Over 20 different extraction and washing conditions ~ 10 years or art. (41 pullouts are shown) Molecular Architecture of the NPC Actual model Alber F. et al. Nature (450) 683-694. 2007 Alber F. et al. Nature (450) 695-700. 2007

7. Interaction Map of Histone Deacetylaces Joshi et al. Molecular Systems Biology 9:672

8. Sowa et al., Cell 2009 Protein Complexes – specific/non-specific binding

9. Choi et al., Nature Methods 2010

10. Tackett et al. JPR 2005 Protein Complexes – specific/non-specific binding

11. M/Z Peptides Fragments Fragmentation Proteolytic Peptides Enzymatic Digestion Protein Complex Chemical Cross-Linking MS MS/MS Isolation Cross-Linked Protein Complex Interaction Partners by Chemical Cross-Linking

12. Protein Crosslinking by Formaldehyde ~1% w/v Fal 20 – 60 min ~0.3% w/v Fal 5 – 20 min 1/100 the volume LaCava

13. Protein Crosslinking by Formaldehyde RED: Formaldehyde crosslinking BLACK: No crosslinking SCORE: Log Ion Current / Log protein abundance

14. M/Z Peptides Fragments Fragmentation Proteolytic Peptides Enzymatic Digestion Protein Complex Chemical Cross-Linking MS MS/MS Isolation Cross-Linked Protein Complex Interaction Sites by Chemical Cross-Linking

15. Cross-linking protein n peptides with reactive groups (n-1)n/2 potential ways to cross-link peptides pairwise + many additional uninformative forms Protein A + IgG heavy chain 990 possible peptide pairs Yeast NPC ˜ 10 6 possible peptide pairs

16. Cross-linking Mass spectrometers have a limited dynamic range and it therefore important to limit the number of possible reactions not to dilute the cross-linked peptides. For identification of a cross-linked peptide pair, both peptides have to be sufficiently long and required to give informative fragmentation. High mass accuracy MS/MS is recommended because the spectrum will be a mixture of fragment ions from two peptides. Because the cross-linked peptides are often large, CAD is not ideal, but instead ETD is recommended.

17. Cloning nanobodies for GFP pullouts Atypical heavy chain-only IgG antibody produced in camelid family – retain high affinity for antigen without light chain Aimed to clone individual single-domain VHH antibodies against GFP – only ~15 kDa, can be recombinantly expressed, used as bait for pullouts, etc. To identify full repertoire, will identify GFP binders through combination of high-throughput DNA sequencing and mass spectrometry VHH clone for recombinant expression

18. Cloning llamabodies for GFP pullouts

19. Identifying full-length sequences from peptides

20. Sequence diversity of 26 verified anti-GFP nanobodies Of ~200 positive sequence hits, 44 high confidence clones were synthesized and tested for expression and GFP binding: 26 were confirmed GFP binders. Sequences have characteristic conserved VHH residues, but significant diversity in CDR regions. FR1 CDR1FR2 CDR2CDR3FR3FR4

21. HIV-1 gp120 Lipid Bilayer gp41 MA CA NC PR IN RT RNA Particle Genome env rev vpu tat nef 3’ LTR 5’ LTR vif gag pol vpr CAMANC p6 PRRTIN gp41gp120 9,200 nucleotides

22. Digestion & Ligation R7/3 + Kan r PmeI Site Kan r Random insertion of 5 amino acids (PmeI) within specific viral coding region Random Insertion of 5 Amino Acids in Proviral DNA Clone

23. Fitness Landscape of Targeted Viral Segment Day 1 Day 3 Day 6

24. Specific and Non-Specific Interactors 3xFLAG Tagged HIV-1WT HIV-1 Infection LightHeavy ( 13 C labeled Lys, Arg) 1:1 Mix Immunoisolation MS I-DIRT = Isotopic Differentiation of Interactions as Random or Targeted LysArg (+6 daltons) Modified from Tackett AJ et al., J Proteome Res. (2005) 4, 1752-6.

25. Specific and Non-Specific Interactors Env-3xFLAGVif-3xFLAG

26. 300 nm 3 nm Limitation of Light Microscopy

27. Fluorescent Imaging with One Nanometer Accuracy (FIONA) Yildiz et al, Science 2003. X axis Y axis CCD image of a single Cy3 molecule: Width ~ 250nm Center is localized within width/(S/N) (S/N) 2 ~ N N = total # photon (for N ~ 10 4 center within ~ 1.3 nm) Paul Selvin

28. Limitation of Light Microscopy 3 nm

29. Limitation of Light Microscopy 3 nm

30. Limitation of Light Microscopy 3 nm

31. Limitation of Light Microscopy 3 nm

32. Limitation of Light Microscopy 20 nm

33. Super-Resolution Localization Microscopy Huang, Annu. Rev. Biochem, 2009 Bates, 2007 Science STORM: STochastic Optical Reconstruction Microscopy Using doubly labeled (Cy3-Cy5) Ab Betzig, 2006 Science PALM: PhotoActivation Localization Microscopy Using fluorescence proteins (mEOS, etc) Using two lasers for interchangeable activation and excitation of probes

34. Molecular Organization of the Intercalated Disc Saffitz, Heart Rhythm (2009)

35. Molecular Organization of the Intercalated Disc Connexin43 (Cx43) Gap junctions Plakophilin-2 (PKP2) Desmosome What is the interaction map of ID proteins? Agullo-Pascual E, Reid DA, Keegan S, Sidhu M, Fenyö D, Rothenberg E, Delmar M. "Super-resolution fluorescence microscopy of the cardiac connexome reveals plakophilin-2 inside the connexin43 plaque“, Cardiovasc Res. 2013

36. Regular Microscopy v. Super-Resolution Cx43 PKP2

37. Cx43 PKP2 Regular Microscopy v. Super-Resolution

38. Cx43 PKP2 Regular Microscopy v. Super-Resolution

39. What Do We Mean by Colocalization?

40. Characterization of Cx43 Clusters Two distinct size populations corresponding to hemi- channels and full channels. Predominantly circular Scale =200 nm

41. Cx43-PKP2 Overlap Analysis A correlation between overlap and Cx43 cluster area 100% overlap 50% overlap Cx43

42. Effect AnkG Silencing on Cx43 AnkG silencing results in increase of Cx43 cluster size and loss of circularity. AnkG Sil 100% overlap 50% overlap

43. Monte-Carlo Simulations

44. Experiment Simulation Experiment Simulation Cx43 PKP2

45. Is the Observed Overlap Random? UntreatedAnkG Silencing Cx43 Area Colocalization Area Cx43 Area Colocalization Area UntreatedAnkG Silencing Uniform Non-uniform Experiment

46. Proteomics Informatics – Protein Characterization II: Protein Interactions (Week 11)