Molecular & Cellular Proteomics 8: , 2009.

Transcription

1 Felix S. Oppermann, Florian Gnad, Jesper V. Olsen, Renate Hornberger, Zolta n Greff, Gyorgy Keri, Matthias Mann, and Henrik Daub. Molecular & Cellular Proteomics 8: , Presented by Yi-Ting Wang Coach Professor : Dr. Kay-Hooi Khoo Sit-in Professor: Dr. Chun-Hung Lin Date:

2 The Characteristics and Significance of Protein Kinase Reversible protein phosphorylation represents the most common type of post-translational modification (PTM) in eukaryotic organisms. Protein kinases : > 500 members in human proteome ATP Protein ADP Protein Kinasekinase The key elements in phosphorylation-based signal transmission. Malignant transformation and tumor development Protein Ser Thr Tyr Protein Ser Thr Tyr P P i Protein Phosphatase Nat. Rev. Cancer. 2008, 8, Nat. Rev. Mol. Cell Biol. 2007, 8,

3 Protein kinases as Cancer Markers for Diagnostics and Therapeutics US FDA approved anticancer drugs targeting protein kinases a) Proteomics Clin. Appl. 2007, 1, Therefore, the study of protein kinases and the phosphorylation is emerged 3

4 Analytical Challenge: In Protein Kinases and It s Phosphorylation Event Low abundance (contamination from presence of other high abundant proteins) Heterogeneous (low substoichiomrtric degree of phosphorylation) Dynamic, spatial change Labile (pser < pthr < ptyr) Consequently, phosphopeptide isolation methods Lower ionization efficiency have proven to be essential. 4

5 Strategies for Phospho-specific Enrichment 1. Only py antibody has specific 2. Lower IP yield Low recovery Phosphoproteomics Method Is Not Sufficient for Kinase Proteins 5

6 Kinase Proteins Enrichment Methods are Required To date, the only enrichment method is the immobilized small molecule inhibitors for affinity capture protein kinases. Exhibit high non-selectivity within the kinase superfamily. Exhibit high selectivity only for kinase superfamily but not for other cellular proteins Kinase Inhibitor Resin Protein Kinase 6

7 Kinase inhibitors as ligand Targeted protein kinase Number of protein kinase ID. SB p38 kinase 12 imatinib, nilotinib, dasatinib bisindolylmaleim ide X, AX14596, SU6668, purvalanol B and VI16832 bisindolylmaleim ide X, AX14596, purvalanol B and PP58 Kinobead kit (7 inhibitors) Kinase-selective Proteomics: BCR-ABL kinase No specificity No specificity a Brief Overview These methods take the No advantages 219 of specificity combining the kinase inhibitors Sample COS-7 and HeLa cells Reference Proc. Natl. Acad. Sci. U.S.A.. (2003) 33 CML patients Blood. (2007) ~183 HeLa S3 cells Jurkat T, A549 cells and HCT- 116 cells 14 human and rodent cell lines and tissues Molecular Cell. (2008) Molecular & Cellular Proteomics (2007) Nature Biotechnology (2007) 7

8 Objective Quantitatively compared the selection efficiency of different kinase inhibitors Example: Immobilized pyrido[2,3-d]pyrimidine-based inhibitors to understand their proteome-wide kinase binding properties 8

9 pyrido[2,3-d]pyrimidine-based Inhibitors PP58 exhibited high potency and non-selectivity for a subset of protein kinases comprising about 25% of the human kinome. Mol. Cell. Proteomics 3, , 2004 They have recently introduced compound with norbornyl moieties (designated VI16832) at the N8 position for enrichment. Mol. Cell, , The actual effect of the N8 substituent has not been systematically analyzed. In the N8 position : With norbornyl moiety With a smaller ethyl moiety With the cyclopentyl moiety Immobilized all three compounds through their primary amino groups 9

10 Schematic of Kinase Proteins Affinity Enrichment 1. Protein mixture/ cell lysate Affinity Column Resin Bead Protein Kinase Kinase inhibitor 2. Unbound proteins Lithium dodecyl sulfate + 3. Elute by detergent and reduction reagent 10

11 Comparative Target Profiling for Kinase-selective Pre-fractionation Reagents The elution fractions from either kinase inhibitor resins or from a control resin (Ctrl) of ECH 11 Sepharose devoid of immobilized ligand were analyzed by SDS PAGE and silver staining.

12 Introduction of SILAC Method SILAC- stable isotope labeling by amino acids in cell culture Mass shift The Mass will be increased after culture in heavy medium. And, the chemical property should not be change. 12 Journal of Proteome Research 2003, 2,

13 Introduction of SILAC Method cont. O O H 2 N CH C OH H 2 N CH C OH CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 NH 2 NH 2 Lys4 Lys8 O O H 2 N CH C OH H 2 N CH C OH CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 NH NH C NH C NH Nature Protocols 6, , 2011 NH 2 Arg6 NH 2 Arg10 13

14 Comparison of Inhibitor Resins by Quantitative Chemical Proteomics Arg 0 /Lys 0 Arg 6 /Lys 4 Arg 10 /Lys 8 14

15 Quantitative Result of Protein Kinases Enriched from VI16743 and VI16741 Ligands VI16743 > VI16741 More then 130 protein kinases are identified and quantified 15

16 Quantitative Result of Protein Kinases Enriched from VI16732 and VI16741 Ligands VI16732 > VI

17 Quantitative Result of Protein Kinases Enriched from VI16732 and VI16743 Ligands VI16732 > VI16743 Overall, VI16732 has better selectivity of kinase proteins due to the bulky structure 17

18 The Quantitative MS Results are Validated by Western Blotting Analysis More then 130 protein kinases are identified and quantified. More than 250 non-protein kinase (non-specific binding). The outcome of western blotting was found in excellent agreement with the MS results 18

19 MS Spectra for SILAC Quantitation Analysis Relative ion intensities changed accordingly in replicate experiments. 19

20 Reproducibility of the SILAC- Based Quantification Independent experimental ratios for VI16743 versus VI16741 as well as for VI16832 versus VI16741 resin binding were similar, demonstrating the accuracy and reliability 20 of the quantitative MS approach.

21 Gene Ontology Analysis of Non-protein Kinases Enriched with Immobilized Kinase Inhibitors Many non-kinase proteins are annotate to protein kinase binding or kinase activity which resulted from the co-purification of protein kinase as well as other regulatory kinase subunits. The enrichment for nucleotidedependent enzymes. These proteins would direct interact with purine-like pharmacophore 21 %/100

22 Comparative Kinase Expression Analysis in Different Cancer Cell Lines MV4 11, leukemia HCT116, colon carcinoma 435S, melanoma-derived cells To monitor cell-type specific differences of kinase expression profiles. In total, more than 170 protein kinases are identified and quantified in three cell lines. The number of protein kinases enriched in a single VI16832 is comparable to seven distinct affinity ligands. 22 Compare to Molecular Cell. (2008)

23 Heat Map to Visualize Quantified Protein Kinases The highest expression in any of the three cell lines set to 1. It s easy to see kinase proteins express level in three kinds of cancer cells by color intensity. 23 Partial list of quantified protein kinases

24 Summed Relative Expression Values for the Major Kinase Groups and Atypical Kinases These seven major kinase grouping are according to Manning et al. Syk, Tec family kinases The expression in three kinds (Btk, of Tec) and several cancer are similar. members of the Src family (Lyn, Fgr, HCK) 24

25 Western Blotting Validation of Three Cancer Cells -in Normal Culture Medium and without Kinase Enrichment Immunoblotting results were found in good agreement with the measured SILAC ratios. These results demonstrate the affinity enrichment can represent the real kinase protein expression 25

26 Large-scale Phosphoproteomics Analysis of Cancer Cell Lines upon Kinase Affinity Enrichment MV4 11 HTC S Affinity purification using VI16832 beads Tryptic in gel digestion Phosphopeptides Enrichment by TiO 2 Identification by LC MS on LTQ Orbitrap This is the largest dataset of site-specific phosphorylation across the kinome and expands the study of protein kinase regulation. 26

27 The Venn Diagrams Show the Cell Line Distribution of the Identified Phosphorylation Sites on Protein Kinases Kinases were classified primarily by sequence comparison of their catalytic domains The Kinase proteins are classified into seven major group and shown in tree diagram Science 298, 1912 (2002) Phosphopeptides identified afrer VI16832 affinity chromatography are marked in the dendrogram and distributed in every kinase group These results indicate the VI16832 ligand has no bias of kinase proteins 27

28 The analysis of three cancer cell lines increased the overall number of identified phosphorylation sites However, without quantitation, the differences of the cancer cells could also be due to run-to-run variability inherent to LC-MS in the data-dependent acquisition mode. 28

29 Distribution of Ser(P), Thr(P), and Tyr(P) in Kinase Protein and Other Phosphoproteins 29

30 Identified phosphorylation sites in the kinase activation loop region of protein kinases Identified phosphorylation site Identified phosphorylation site in this cell and highlighted in yellow Not Identified phosphorylation site in this cell line tripeptide motifs DFG and APE, which define the borders of the activation segment VI16832 mediated enrichment provides an experimental basis to monitor the activation segment for more than 50 kinases per cell line 30

31 Summary VI16832 have better enrichment specificity for kinase proteins which is evaluated by quantitatively comparison method, SILAC. - More than 170 protein kinases are identified and quantified. - By phosphoproteomics survey, about 1200 phosphorylation sites are identified on more than 200 protein kinases. These results demonstrate the practicability of quantitative comparison of kinome profiling. 31

32 Conclusion Combination of kinase-selective enrichment, quantitative MS, and phosphopeptide purification enable the discovery of signal transduction and kinase drug target analysis. 32

33 Thank you for your attention. 33

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36 ATP binding site of p90 ribosomal S6 kinase Science, 2005, 308,