1. Phosphoproteome Analysis by Mass Spectrometry Jau-Song Yu ( 余兆松 ) Department of Cell and Molecular Biology, Institute of Basic Medical Sciences, Medical College of Chang Gung University ( 長庚大學基礎醫學所分子生物學科 )

2. Reversible Phosphorylation of Proteins Protein/Enzyme OH PO 4 Protein kinaseProtein phosphatase Cellular Processes: Metabolism, contractility, membrane transport and secretion, transcription and translation of genes, cell division, fertilization, memory, carcinogenesis, apoptosis, etc. (Ser, Thr or Tyr)

3. The 1992 Nobel Prize in Physiology or Medicine NOBELF Ö RSAMLINGEN KAROLINSKA INSTITUTET THE NOBEL ASSEMBLY AT THE KAROLINSKA INSTITUTE (12 October 1992) The Nobel Assembly at the Karolinska Institute has today decided to award the Nobel Prize in Physiology or Medicine for 1992 jointly to Edmond H. Fischer and Edwin G. Krebs for their discoveries concerning "reversible protein phosphorylation as a biological regulatory mechanism". Summary Thousands of proteins participate in a complex interplay in a cell. They are the tools of the living organism, regulating its reactions and activities. For example, proteins maintain the metabolic flux, dictate growth and cellular division, release hormones, and mediate muscular work. Protein interactions are strictly controlled. One of the most important regulatory mechanisms is reversible protein phosphorylation. This means that enzymes phosphorylate and dephosphorylate proteins. Both these enzymatic processes are in turn regulated, often in several steps, allowing amplification and fine control. The 1992 Nobel Prize in Physiology or Medicine is awarded to the American biochemists Edmond Fischer and Edwin Krebs. They purified and characterized the first enzyme of this type. Their fundamental finding initiated a research area which today is one of the most active and wide-ranging. Reversible protein phosphorylation is responsible for regulation of processes as diverse as mobilization of glucose from glycogen, prevention of transplant rejection by cyclosporin, and development of a cancer form like chronic myeloic leukemia.

4. Phosphoryl groups affect the structure and catalytic activity of proteins Glycogen phosphorylase (Glucose)n + Pi (glucose)n- 1 + glucose 1-phosphate

5. AMP P-Ser 14 Glucose PLP Regulation of glycogen phosphorylase Pyridoxal phosphate (PLP) Un-P: 20 aa (+) residues at its N terminus Interact with multiple acidic aa P-Ser14: interferes this interaction, more active conformation

6. The 2001 Nobel Prize in Physiology or Medicine 8 October 2001 The Nobel Assembly at Karolinska Institutet has today decided to award The Nobel Prize in Physiology or Medicine for 2001 jointly to Leland H. Hartwell, R. Timothy (Tim) Hunt and Paul M. Nurse for their discoveries of "key regulators of the cell cycle" Summary All organisms consist of cells that multiply through cell division. An adult human being has approximately 100 000 billion cells, all originating from a single cell, the fertilized egg cell. In adults there is also an enormous number of continuously dividing cells replacing those dying. Before a cell can divide it has to grow in size, duplicate its chromosomes and separate the chromosomes for exact distribution between the two daughter cells. These different processes are coordinated in the cell cycle. This year's Nobel Laureates in Physiology or Medicine have made seminal discoveries concerning the control of the cell cycle. They have identified key molecules that regulate the cell cycle in all eukaryotic organisms, including yeasts, plants, animals and human. These fundamental discoveries have a great impact on all aspects of cell growth. Defects in cell cycle control may lead to the type of chromosome alterations seen in cancer cells. This may in the long term open new possibilities for cancer treatment.

8. Kinase distribution by major groups in human and model systems SCIENCE, 298, 1912-34 (2002) The Protein Kinase Complement of the Human Genome G. Manning, 1 * D. B. Whyte, 1 R. Martinez, 1 T. Hunter, 2 S. Sudarsanam 1,3

9. Strategy for kinase activity detection in cells

10. Kinase assay in immunoprecipitate (IP) Cells *homogenization (10-cm dish/0.5 ml lysis buffer) *centrifugation (12000~15000 rpm, 15 min, 4 o C) Supernatants *protein concentration determination *1 mg protein/0.5 ml extracts *add Ab against specific kinase (5  g) *incubation (1 h, 4 o C) *add protein A/G-S4B (50% v/v, 25  l, shaking) *centrifugation (6000 rpm, 1min, 4 o C) *wash/cfg 3 times in Buffer B Immunoprecipitates *suspended in 20  l Buffer A *substrate (5-10  g), [  - 32 P]ATP.Mg 2+ (0.2-20 mM) *shaking for 10-30 min at RT *adding SDS-sample buffer SDS-PAGE Autoradiography Lysis buffer-----10 mM Tris-HCl at pH 7.4, 2 mM EDTA, 1 mM EGTA, 1% Triton X-100, 1 mM benzamidine, 1 mM phenylmethylsulfonyl fluoride, 0.5 mg/ml aprotinin Buffer A --- 20 mM Tris-HCl at pH 7.0, 0.5 mM dithiothreitol Buffer B --- 0.5 M NaCl in buffer A (quantitative method) cfg

11. JNK activity assay in IP (Chan et al., 2000)

12. Kinase assay by immunoblotting with phospho-specific Ab (Qualitative to semi-quantitative method) JNK1 p-JNK1 C CL P 0 0.5 1 1.5 2 3 4 Time post PDT (hr) p-JNK2 (Hsieh et al., 2003)

13. Determination of protein phosphorylation sites Protein/Enzyme OH PO 4 Protein kinaseProtein phosphatase (Ser, Thr or Tyr?) (What a.a. and where?)

14. Mark O. Collins, Lu Yu and Jyoti S. Choudhary: Analysis of protein phosphorylation on a proteome-scale. Proteomics (7) 2751 – 2768, 2007 Edman Degradation ( 32 P-release) Strategy of Phosphorylation Site Analysis Phosphoamino acid analysis (Ser, Thr or Tyr?)

15. 1 2 3 4 5 6 32 P-labeled proteins in IP fractions from A431 cells Phosphoamino acid analysis (1) (3) (7-10 days) (16 hrs)

16. Edman Degradation ( 32 P-release)

24. Modern Strategy of Phosphoproteome Analysis B. C. A. Mark O. Collins, Lu Yu and Jyoti S. Choudhary: Analysis of protein phosphorylation on a proteome-scale. Proteomics (7) 2751 – 2768, 2007 efficiencyaccuracyScale Edman degadation lowexcellent Single protein MS analysishighgoodSystemic

25. SKRSTMVGTPYC Y11 y10 y9 y8 y7 y6 y5 y4 y3 y2 y1 b1 b2 b3 b4 b5 b6 b7 b8 b9 b10 b11 SKRSTMVGTPYC Y11 y10 y9 y8 y7 y6 y5 y4 y3 y2 y1 b1 b2 b3 b4 b5 b6 b7 b8 b9 b10 b11 P

26. 1. Ionization 2. MS 2 MS 3 -98  -elimination (NaOH…)

27. H-SKRSpTMVGTPYC –OH 1409.608 -98Da MS

28. H-SKRSTMVGTPYC -OH 1311.572 b5 TOF/TOF Intens. [a.u.] 129.022 381.919 215.988 69.952 b5 542.053 283.908 431.059 1262.511 157.001 344.017 1206.757 507.080 477.012 1119.588 597.040 580.035 744.183 495.014 673.110 324.969 930.523 782.079 0.0 0.2 0.4 0.6 0.8 1.0 1.2 4 x10 20040060080010001200 m/z 1311.572 372.919

29. 129.022 381.919 b2 215.988 69.952 b5 542.053 283.908 431.059 1262.511 157.001 344.017 1206.757 1119.588 597.040 744.183 b6 673.110 324.969 930.523 782.079 0.0 0.2 0.4 0.6 0.8 1.0 1.2 4 x10 20040060080010001200 m/z 1311.572 H-SKRSpTMVGTPYC -OH 1409.560 H-SKRSTMVGTPYC -OH 1311.572 b5 1311.582 542.175 b 3 372.134 1409.560 b5 640.155b4 459.149 b7 870.250 129.081 216.108 b6 771.162 930.349 1010.307 284.032 0 1 2 3 4 5 6 4 x10 Intens. [a.u.] 200400600800100012001400 m/z b2 b9 1028.189 b8 927.360 - b3 372.919 b4 458.919 METHOD 1 1329.634

30. Proteomics 2008, 8, 4416–4432

31. Systematic analysis of protein phosphorylation by MS Temporal analysis of phosphotyrosine-dependent signaling networks by quantitative proteomics Blagoy Blagoev, Shao-En Ong, Irina Kratchmarova & Matthias Mann Nature Biotechnology 22 1139-1145 (2004 ) Mass spectrometry and data analysis. Mass spectrometric analyses were done with nanoscale LC-mass spectrometry (LC-MS) and LC-tandem mass spectrometry and a quadrupole time-of-flight instrument (QSTAR-Pulsar, ABIMDS-SCIEX) with sample introduction with a 96-well autosampler (Agilent HP1100). MS MS+6 MS+10

32. Upregulated proteins

33. Downregulated proteins

34. Figure 2. Western blot analysis of selected EGFR effectors. HeLa cells were stimulated with EGF for the indicated time intervals, matching the proteomics experiments. 1.1--7.1--15.4--2.1 3.5--3.6--2.7--1.7 23.6--20.5--8.8--3.7 34--28.6--17.9--2.8 1.8--8.6--6.1--1.7 1.0--2.1--4.0--1.2 29.7--11--3.2--2.8 9.3--10--2.7--1.7 0.44--0.57--0.66--0.76 42--60--18.8--7.1 39--40--31.5--17.8 Fold activation

35. Receptor internalization Ras-MAPK pathways Actin remodeling Novel proteins

36. Quantitative proteome analysis of the P-STM antibody-recognizable phosphorylation site on lamins A/C in mitotic HeLa S3 cells (Yu et al. Biochem J, 1998)

37. *Department of Cell and Molecular Biology, Institute of Basic Medicine, Chang Gung University, Tao-Yuan, Taiwan, R.O.C., and.Department of Life Sciences, National Tsing Hua University, Hsinchu, Taiwan, R.O.C. Yang, Fong, Yu and Liu (1987) J. Biol. Chem. 262, 7034-40

38. Edman Degradation ( 32 P-release)

39. Immunoblot analysis of auto-kinase during the activation and inactivation processes with a phospho-specific antibody (P-STM Ab) against the identified phosphorylation-site sequence (Yu et al., Biochem. J. 1998)

40. Anti-phosphopeptide antibody, P-STM as a novel tool for detecting mitotic phosphoproteins: Identification of lamins A and C as two major targets Tsai et al. J. Cell. Biochem. 94, 967–981 (2005)

41. 5, 19, 22 199 390, 392 416, 403, 404 480 525 Proc Natl Acad Sci U S A. 2004 Aug 17;101(33) Eur J Cell Biol. 1993 Dec;62(2):237-47 Eur J Cell Biol. 1993 Dec;62(2):237-47. Cell. 1990 May 18;61(4):579-89. J Cell Biol. 1996 Dec;135(6 Pt 1):1441-55 EMBO J. 2002 Apr 15;21(8):1967-77 Eur J Cell Biol. 1993 Dec;62(2):237-47 Cell. 1990 May 18;61(4):579-89 Eur J Cell Biol. 1993 Dec;62(2):237-47. ＊ ＊ It’s not easy to assess the dynamic change of specific phosphorylation site on lamin A/C during cell cycle 12 SGAQASS 19 TPL 22 SPTR 389 LSP 392 SPTSQR SKRS[pT 402 ] MVGTPYC

42. Cell. 1990 May 18;61(4):579-89. Mutations of phosphorylation sites in lamin A that prevent nuclear lamina disassembly in mitosis. Heald RHeald R, McKeon F.McKeon F Department of Cellular and Molecular Physiology, Harvard Medical School, Boston, Massachusetts 02115. The nuclear envelope is a dynamic structure that completely disassembles in response to MPF/cdc2 activity in mitosis. A key feature of this process is the hyperphosphorylation of the major structural proteins of the envelope, the nuclear lamins A, B, and C. Two highly conserved serine residues of the lamin protein (Ser-22 and Ser-392 of lamins A and C) are symmetrically positioned 5 amino acids from the ends of the large alpha-helical domain and are shown in the accompanying paper by Ward and Kirschner to be among four sites phosphorylated during nuclear envelope breakdown. Mutations in Ser-22 and Ser-392 that prevent phosphorylation at these sites block the disassembly of the nuclear lamina during mitosis. We propose a model for the regulation of lamin assembly in which phosphorylation just outside the ends of the alpha-helical domain controls the assembly dynamics of the lamin coiled-coil dimers.

43. Stable isotope labeling by amino acids in cell culture, SILAC, as a simple and accurate approach to expression proteomics. Ong, S.E. Blagoev B, Kratchmarova I, Kristensen DB, Steen H, Pandey A, Mann M. Department of Proteomics and Signal Transduction, Max Planck Institute for Biochemistry, Germany. Mol. Cell. Proteomics 1, 376–386 (2002).NATURE PROTOCOLS 1, 2650 (2006)

45. PNAS USA 100, 15434–15439 (2003) Fig. 2. Optimization of adsorption and elution conditions for a functional p38 inhibitor matrix. Fig. 1. Identification of a p38 inhibitor analogue suitable for immobilization.

46. Fig. 3. Efficient affinity purification of protein kinases specifically targeted by immobilized p38 inhibitor. HeLa whole cell lysate was subjected to PI 51 affinity chromatography, and the bound proteins were eluted with a combination of ATP and free PI 51. 16-benzyldimethyl-n-hexadecylammonium chloride (16-BAC)

47. Fig. 5. In vitro characterization of protein kinases inhibited by SB 203580. Fig. 6. Structural determinants of SB 203580 sensitivity.