1. Lecture 10. Proteomics II **Lewis TS, Hunt JB, Aveline LD, Jonscher KR, Louie DF, Yeh JM, Nahreini TS, Resing KA, Ahn NG. 2000. Identification of novel MAP kinase pathway signaling targets by functional proteomics and mass spectrometry. Mol Cell 6:1343-1354 Zhu H, Bilgin M, Bangham R, Hall D, Casamayor A, Bertone P, Lan N, Jansen R, Bidlingmaier S, Houfek T, Mitchell T, Miller P, Dean RA, Gerstein M, Snyder M. 2001. Global analysis of protein activities using proteome chips. Science. 293:2101-2105.

2. Paper 1. Two parts: 1. 2D-gels to Display the Proteome and Potential Erk1/2 Targets 2. MS to Identify the Erk1/2 Targets Lewis et al. 2000. Identification of novel MAP kinase pathway signaling targets by functional proteomics and mass spectrometry. Mol Cell 6:1343-1354

3. The Mitogen Activated Protein Kinase (MAPKK) Family MAPKKK MAPKK MAPK RafMKK1MKK4 MEK1/2 JEK1/2MKK6 ERK1/2JNK1/2p38 Growth Differentiation Apoptosis Stress Responses

4. Phorbol Ester (PMA) ras raf MEK1/2 Erk1/2 Protein Kinase C (PKC) Differentiation K562 cells To Megakaryocytes (Platelets)

5. Part 1. 2D Gels to Compare the Proteome in PMA treated K562 cells 3500 proteins resolved: detection from 5000-10 7 copies per cell 91 proteins found different in PMS treated cells (30 down, 61 up): magnitude change >1.5-fold; reproducible in 3 gels from at least 2 experiments

6. Kinetic Analysis of PMA-dependent changes in expression Down Up Mobility Altered

7. Cluster Analysis of the 91 Changes Reveals 4 General Classes

8. Phorbol Ester (PMA) ras raf MEK1/2 Erk1/2 Protein Kinase C (PKC) Differentiation K562 cells To Megakaryocytes (Platelets) U0126 Question: Are these proteins regulated by Erks or PKC? 1. Pharmacological Inhibitor Should Inhibit Changes

9. 1. Pharmacological Inhibitor Should Inhibit Changes

10. No Phorbol Ester (PMA) ras raf MEK1/2* Erk1/2 Differentiation K562 cells To Megakaryocytes (Platelets) MEK1 Dominant Positive Mutation Question: Are these proteins regulated by Erks or PKC? 2. Dominant Signaling Mutations Should Cause Changes in Absence of PMA

11. 2. Dominant Signaling Mutations Should Cause Changes in Absence of PMA

12. Correspondence Between U0126 Inhibition and MKK1* Stimulation Conclusion: 66% of Changes Are DIRECT Targets of Erk1/2

13. Peptide Mixture MALDI (Matrix Assisted Laser Desorption Ionization) Mass Spec Used to Fingerprint the 91 proteins

14. HPLC coupled with nanospray MS Used to Confirm Identity of the Proteins -Result: 41 proteins account for the 91 Changes -25 are Direct Targets of Erks -20/25 Represent newly Defined Targets

16. Product:Precursor Relationships

17. Paper 2. Protein Chips to Study Protein:Protein or Protein:Lipid Interactions Zhu H, et al 2001 Global analysis of protein activities using proteome chips. Science. 293:2101-2105.

18. Yeast Proteome on a Single Glass Slide: 5800 GST Proteins Purified and Placed on the Chip Probed with anti-GST as a CONTROL

19. Finding All Protein Partners for Calmodulin: 14/39 Contain a Conserved Calmodulin Motif Biotinylated Calmodulin Used to Probe Chip

20. Identification of Lipid Binding Proteins

21. PI3 Interacting Proteins Specific fir PI3 Bind PI3 and PC

22. Conventional Techniques Confirm Lipid Binding by Proteins Identified with the CHIP