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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
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Content
Introduction
Methods
Results
Discussion
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Einführung
Cancer:
•A major cause of death
•Lung Cancer (85% NSCLC)
Cancer establishment:
•6 hallmarks of cancer
•No exact pathway
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Einführung
Biomarkers:
•Since antiquity
•DNA biomarker
•Protein biomarker
– e.g. Kinase
Discovery:
•Needle in the haystack
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Einführung
Aim:
•Kinase as biomarker
To-Do:
•Healthy vs. Cancer
•Kinase enrichment
•Quantification
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Methods
Workflow:
• Tissue lysis
• Affinity pulldown
• LC-MS/MS
• Quantification
• Identification
• Statistical analysis
Kinome enrichment
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Methods
Affinity pulldown:
•Kinobeads
– Kinase inhibitors
Protein Quantification
•Progenesis LC-MS
– Intensity based quantification
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Results
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Results
Quantification method comparison
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Ergebnisse
Reproducibility
•Correlation Coefficient
•All identified kinases
•Kinases with >3 peptides for quantification
•Higher reproducibility in the higher intensity part
Sample Correlation [1/2] Correlation [1/2] >3
15 57.70 86.89
16 85.6 91.8
18 91.3 94.38
21 90 93.91
33 75 85
36 37.2 73.33
56 48 54.35
57 55 75.42
62 81 93.19
65 80 98
86 89 90
87 89 97.66
90 87.44 92.95
97 78.30 90.70
121 53.2 70.96
122 71 90.27
127 87,4 95.38
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Results
Data Evaluation
•Progenesis data
•VSN normalized data
•QQ-Plots
– Straight line indicates normal distribution
•Scatter plots
Progenesis
VSN data
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Results
Progenesis VSN
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Results
PCA Analysis
•Progenesis data
A: Proteins B: Cancer Control
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Results
PCA Analysis
•VSN data
A: Proteins B: Cancer Control
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Results
Kinase Clustering
Progenesis VSN
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Results
Ingenuity Pathway
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Results
Kinase profiling
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Results
Patient profiling
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Discussion
Quantification
•Only „Chemical labeling“ or label-free
Sample Exclusion
•Cancer Sample 33
•Cancer Sample 36
•Control Sample 62
50 kD
C 33E 33C 36E 36C 56
C 33E 33C 36E 36C 56E 56HOP 62E 62
75 kD
50 kD
Sample Correlation [1/2] Correlation [1/2] >3
36 37.2 73.33
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Discussion
Kinase profiling
•Overall expression
Patient profiling
•Stratification
IPA
•Pathway prediction
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Discussion
Sample type
•Biopsy
•Blood
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Outlook
Further screening
Not yet diseased
Smokers vs. Non-smokers
Therapy context
Blood sample
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Thank you for your attention!