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

Content

Introduction

Methods

Results

Discussion

Einführung

Cancer:

•A major cause of death

•Lung Cancer (85% NSCLC)

Cancer establishment:

•6 hallmarks of cancer

•No exact pathway

Einführung

Biomarkers:

•Since antiquity

•DNA biomarker

•Protein biomarker

– e.g. Kinase

Discovery:

•Needle in the haystack

Einführung

Aim:

•Kinase as biomarker

To-Do:

•Healthy vs. Cancer

•Kinase enrichment

•Quantification

Methods

Workflow:

• Tissue lysis

• Affinity pulldown

• LC-MS/MS

• Quantification

• Identification

• Statistical analysis

Kinome enrichment

Methods

Affinity pulldown:

•Kinobeads

– Kinase inhibitors

Protein Quantification

•Progenesis LC-MS

– Intensity based quantification

Results

Results

Quantification method comparison

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

Results

Data Evaluation

•Progenesis data

•VSN normalized data

•QQ-Plots

– Straight line indicates normal distribution

•Scatter plots

Progenesis

VSN data

Results

Progenesis VSN

Results

PCA Analysis

•Progenesis data

A: Proteins B: Cancer Control

Results

PCA Analysis

•VSN data

A: Proteins B: Cancer Control

Results

Kinase Clustering

Progenesis VSN

Results

Ingenuity Pathway

Results

Kinase profiling

Results

Patient profiling

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

Discussion

Kinase profiling

•Overall expression

Patient profiling

•Stratification

IPA

•Pathway prediction

Discussion

Sample type

•Biopsy

•Blood

Outlook

Further screening

Not yet diseased

Smokers vs. Non-smokers

Therapy context

Blood sample

Thank you for your attention!