1. Technische Universität München Cancer biomarker discovery by chemical and quantitative proteomics Sebastian Berger, B. Sc., 3010425 Center of Life and Food Sciences Weihenstephan Chair of Proteomics and Bioanalytics Master Kolloquium

2. Content  Introduction  Methods  Results  Discussion

3. Einführung  Cancer: A major cause of death Lung Cancer (85% NSCLC)  Cancer establishment: 6 hallmarks of cancer No exact pathway

4. Einführung  Biomarkers: Since antiquity DNA biomarker Protein biomarker –e.g. Kinase  Discovery: Needle in the haystack

5. Einführung  Aim: Kinase as biomarker  To-Do: Healthy vs. Cancer Kinase enrichment Quantification

6. Methods  Workflow: Tissue lysis Affinity pulldown LC-MS/MS Quantification Identification Statistical analysis Kinome enrichment

7. Methods  Affinity pulldown: Kinobeads –Kinase inhibitors  Protein Quantification Progenesis LC-MS –Intensity based quantification

8. Results

9.  Quantification method comparison

10. Ergebnisse  Reproducibility Correlation Coefficient All identified kinases Kinases with >3 peptides for quantification Higher reproducibility in the higher intensity part SampleCorrelation [1/2]Correlation [1/2] >3 1557.7086.89 1685.691.8 1891.394.38 219093.91 337585 3637.273.33 564854.35 575575.42 628193.19 658098 868990 8789 97.66 9087.44 92.95 9778.3090.70 12153.270.96 1227190.27 12787,4 95.38

11. Results  Data Evaluation Progenesis data VSN normalized data QQ-Plots –Straight line indicates normal distribution Scatter plots Progenesis VSN data

12. Results Progenesis VSN

13. Results  PCA Analysis Progenesis data

14. Results  PCA Analysis VSN data

15. Results  Kinase Clustering ProgenesisVSN

16. Results  Ingenuity Pathway

17. Results  Kinase profiling

18. Results  Patient profiling

19. Discussion  Quantification Only „Chemical labeling“ or label-free  Sample Exclusion Cancer Sample 33 Cancer Sample 36 Control Sample 62 50 kD C 33 E 33C 36E 36 C 56 C 33 E 33C 36 E 36 C 56 E 56 HOP 62 E 62 75 kD 50 kD SampleCorrelation [1/2]Correlation [1/2] >3 3637.273.33

20. Discussion  Kinase profiling Overall expression  Patient profiling Stratification  IPA Pathway prediction

21. Discussion  Sample type Biopsy Blood

22. Outlook  Further screening  Not yet diseased  Smokers vs. Non- smokers  Therapy context  Blood sample

23. Thank you for your attention!