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A biochemists' tool kit to study the battlefront up close

A biochemists' tool kit to study the battlefront up close. (the techniques used to study protein--protein interactions). Proteomics. Proteome = All the prote ins expressed by a specific gen ome or tissue. Gene sequence (DNA).

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A biochemists' tool kit to study the battlefront up close

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  1. A biochemists' tool kit to study the battlefront up close (the techniques used to study protein--protein interactions)

  2. Proteomics Proteome = All the proteins expressed by a specific genome or tissue Gene sequence (DNA) gtc tac ctc cag acc tcc ttg aaa tac aat att ctc cca gaa aag gaa gag K C R V N C F K L S I L P E K E E Protein sequence But, genes can be translated several ways to create >1 protein per gene And then, proteins can be modified to create >1 form of the protein per sequence

  3. How a MALDI mass spectrometer works Ion counts 1000 2000 3000 m/z

  4. Mass spectrometer schematics Vacuum envelope Sample in InletSystem Ion Source Mass Analyzer Detector Data System Inlet systems: • Simple vacuum lock • HPLC • GC Ion sources: • Electrospray (ESI) • MALDI • FAB/LSIMS • Electron ionization (EI) Mass spectrum out Mass analyzers: • Quadrupole • Time-of-flight • Ion trap • Magnetic sector • FTMS

  5. Targeted Proteomics Fish with “bait” (interesting protein) Pull-out interacting proteins Excise bands, Identify proteins

  6. CellularProteomics Cell in state A Cell in state B Compare 2D gels Digest all proteins, LC-MS/MS

  7. The human interactome (500 baits)

  8. LRP1 may provide the connection between damage inside and outside neurons in Alzheimer’s TNFTNPVYATLY PLCγ-1 Sos-1 TNFTNPVpYATLY Histone Deacetylase 6 PI3 kinase p110 & GAP1 SYP PI3 kinase p85 CRAF1, Shp1, Shp2 CaM kinase II Src CSK Shc Elongation Factor 1α Crkl Ck1-α Pyrroline – 5 carboxylate reductase Ribosomal S4 Grb2

  9. Heart disease involves too much blood clotting antithrombinIII Thrombin + Thrombomodulin Protein C Activated Protein C

  10. The thrombin-thrombomodulin interaction takes place in the flowing blood Blood Vessel Endothelium Protein C TM Activated Protein C Thrombin-TM Thrombin

  11. Surface plasmon resonance: real-time binding kinetics of interactions in a flowing system q TM Thrombin-TM Thrombin Thrombin Injection Buffer Injection Time (in seconds)

  12. 0 100 200 300 400 The thrombin-thrombomodulin interaction is fast-on, fast-off Thrombin-mAb Thrombin-TM456

  13. The MALDI-Mapping Experiments D H D D D D H D D H H D D D D D D H H D The on-exchange experiment measures solvent accessibility pepsin H D2O D2O D t (long) t (short) H H The off-exchange experiment measures retention of amide deuteration at protein-protein interfaces. D H D H2O pepsin D D D H H H D D H Both on-exchange and off-exchange data are required to distinguish interface protection from conformational changes.

  14. MALDI-Mapping shows that Thrombomodulin changes the active site of thrombin

  15. Conclusions • Mass spectrometry is useful for finding protein-protein interaction binding partners • Our lab is finding new target sites for Alzheimer’s Disease • MALDI-mapping helps find binding sites • Our lab is using MALDI mapping to find activity changes in proteins induced by an interaction partner

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