Quantitative chemical proteomics for cancer characterization:
Gespeichert in:
| 1. Verfasser: | |
|---|---|
| Format: | Abschlussarbeit Buch |
| Sprache: | English |
| Veröffentlicht: |
2012
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| Schlagworte: | |
| Online-Zugang: | kostenfrei https://nbn-resolving.org/urn:nbn:de:bvb:91-diss-20120511-1097813-1-3 Inhaltsverzeichnis |
| Beschreibung: | Zsfassung in dt. Sprache |
| Beschreibung: | IV, 137 S. Ill., graph. Darst. |
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Datensatz im Suchindex
| _version_ | 1804149288375156736 |
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| adam_text | IMAGE 1
TABLE O F CONTENTS
1. GENERAL INTRODUCTION 1
1.1 CANCER AND KINASE IN CANCER 1
1.2 MASS SPECTROMETRY-BASED PROTEOMICS 5 .
1.2.1 SAMPLE PREPARATION 7
1.2.2 MS ANALYSIS 9
1.2.3 DATA PROCESSING 14
1.2.4 MASS SPECTROMETRY-BASED QUANTITATIVE PROTEOMICS 15
1.3 CHEMICAL PROTEOMICS 17
1.4 KINOME PROFILING 19
1.5 AIM AND OUTLINE OF THIS THESIS 20
2. ESTABLISHMENT AND OPTIMIZATION OF KINOBEAD BASED QUANTITATIVE
CHEMICAL PROTEOMICS 21
2.1 INTRODUCTION 21
2.2 MATERIAL AND METHODS 23
2.2.1 CELL CULTURE AND HARVESTING 23
2.2.2 STUDY FOR CELL LYSIS WITH DIFFERENT DETERGENTS 23
2.2.3 STUDY FOR CELL LYSIS WITH DIFFERENT CONCENTRATIONS OF PHOSPHOTASE
INHIBITORS 23
2.2.4 PLACENTA LYSIS 24
2.2.5 AFFINITY PURIFICATION AND PROTEIN DIGESTION 24
2.2.6 IN-SOLUTION PROTEIN DIGESTION 25
2.2.7 IN-GEL PROTEIN DIGESTION 25
2.2.8 DIMETHYL LABELING OF TRYPTIC PEPTIDES 25
2.2.9 STAGE-TIPS PURIFICATION 26
2.2.10 LC-MS/MS ANALYSIS 26
2.2.11 PEPTIDE AND PROTEIN IDENTIFICATION AND QUANTIFICATION 26
2.2.12 STATISTICAL ANALYSIS 28
2.3 RESULT AND DISCUSSION 28
2.3.1 KINOBEAD ESTABLISHMENT 28
2.3.2 DETERGENT SELECTION 30
2.3.3 KINOBEAD CAPACITY 31
2.3.4 CONCENTRATION OF PHOSPHOTASE INHIBITORS 33
I
HTTP://D-NB.INFO/1032222972
IMAGE 2
TABLE O F CONTENTS
2.3.5 COMPARISON OF SPECTRA COUNTING BASED AND INTENSITY BASED
LABEL-FREE QUANTIFICATION. 34
2.3.6 COMPARISON OF THE DIMETHYL LABELING AND INTENSITY BASED LABEL-FREE
QUANTIFICATION 37
2.3.7 VIABILITY OF INTENSITY BASED LABEL-FREE QUANTIFICATION FOR
CHEMICAL PROTEOMICS 39
2.4 CONCLUSION 40
3. QUANTITATIVE CHEMICAL PROTEOMICS REVEALS NEW POTENTIAL DRUG TARGETS
IN HEAD AND NECK CANCER
42
3.1 INTRODUCTION 42
3.2 MATERIAL AND METHODS 44
3.2.1 HNSCC CELL LINES 44
I
3.2.2 CELL CULTURE AND HARVESTING 44
3.2.3 AFFINITY PURIFICATION AND PROTEIN DIGESTION 45
3.2.4 LC-MS/MS ANALYSIS 45
3.2.5 PEPTIDE AND PROTEIN QUANTIFICATION AND IDENTIFICATION 45
3.2.6 STATISTICAL ANALYSIS : 45
3.2.7 WESTERN BLOT ANALYSIS 46
3.2.8 IMMUNOHISTOCHEMISTRY ANALYSIS 46
3.2.9 SAMPLE PREPARATION FOR DNA SEQUENCING : 47
3.2.10 KINASE INHIBITOR TREATMENT 48
3.2.11 SIRNA TREATMENT 48
3.2.12 EPHRIN A1 FC TREATMENT 48
3.3 RESULTS AND DISCUSSION 49
3.3.1 EXPERIMENTAL STRATEGY OF IDENTIFYING DIFFERENTIAL KINASE PROTEIN
EXPRESSION IN HNSCC
CELL LINES 49
3.3.2 TARGETS PROFILING ON HNSCC BY CHEMICAL PROTEOMICS COMBINED WITH
QUANTITATIVE MASS
SPECTROMETRY 50
3.3.3 STATISTIC ANALYSIS 52
3.3.4 VALIDATION OF THE MS DATA BY WESTERN BLOT 54
3.3.5 IMMUNOHISTOCHEMICAL ANALYSIS OF EGFR ON CLINICAL SAMPLE 55
3.3.6 CONFIRMATION OF EGFR CONTRIBUTING TO THE PROLIFERATION OF SOME BUT
NOT ALL HNSCC 56
3.3.7 SYNONYMOUS SUBSTITUTIONS IN EGFR ENCODING SEQUENCE 59
3.3.8 LOSS OF FUNCTION SCREENING BY SIRNA HIGHLIGHTS NEW CANDIDATE DRUG
TARGETS 60
II
IMAGE 3
TABLE O F CONTENTS
3.3.9 TARGET VALIDATION OF EPHA2 62
3.3.10 EPHA2 WAS DIFFERENTIALLY EXPRESSED IN THE CLINICAL SAMPLES 63
3.3.11 COMBINATION TREATMENT BY TARGETING EGFR AND EPHA2 65
3.3.12 PATHWAY ANALYSIS 66
3.3.13 INVESTIGATION OF SRC FAMILY KINASES IN HNSCC 67
3.3.14 EVALUATION OF C-MET AS A TARGET IN HNSCC 69
3.4 CONCLUSION 7 1
4. SYSTEMATIC IDENTIFICATION OF THE HSP90 REGULATED PROTEOME 73
. 4.1 INTRODUCTION 73
4.2 MATERIAL AND METHODS 76
4.2.1 SILAC LABELING AND CELL CULTURE 76
4.2.2 PULSED SILAC 76
4.2.3 DRUG TREATMENT AND HARVESTING 76
4.2.4 GEL BASED SAMPLE PREPARATION FOR FULL PROTEOME 77
4.2.5 AFFINITY PURIFICATION 77
4.2.6 IMMUNOPRECIPITATION AND GA-NHS AFFINITY PURIFICATION 77
4.2.7 LC-MS/MS ANALYSIS 78
4.2.8 PEPTIDE AND PROTEIN QUANTIFICATION AND IDENTIFICATION 78
4.2.9 STATISTIC ANALYSIS 78
4.2.10 GO ENRICHMENT/ PATHWAY ANALYSIS/COMPLEX ANALYSIS 79
4.2.11 IMMUNOBLOT ANALYSIS 79
4.3 RESULTS AND DISCUSSION 80
4.3.1 STRATEGY OF KINOME AND PROTEOME WIDE INVESTIGATION OF HSP90
REGULATION 80
4.3.2 GA REDUCES CELL VIABILITY 8 1
4.3.3 PROTEOME WIDE MASS SPECTROMETRIC IDENTIFICATION AND QUANTIFICATION
OF PROTEIN
CHANGES INDUCED BY GA 82
4.3.4 DATA EVALUATION 83
4.3.5 HIGH QUALITY MAP OF HSP90 REGULATED PROTEINS 85
4.3.6 KINOME-WIDE QUANTITATIVE PROFILING REVEALS NEW POTENTIAL CLIENTS
OF HSP90 86
4.3.7 GO ANALYSIS REVEALS SUBGROUPS OF PROTEINS WITH DIVERSE RESPONSE TO
THE HSP90
INHIBITION 88
III
IMAGE 4
TABLE O F CONTENTS
4.3.8 CANCER SPECIFIC PATHWAYS AND NETWORKS INVOLVED IN DIFFERENT
CANCERS 91
4.3.9 CO-REGULATION IN PROTEIN COMPLEX INDUCED BY GA 92
4.3.10 PHARMACOLOGICAL INTERVENTION WITH HSP90 REGULATED PATHWAYS 94
4.3.11 PROTEIN TURNOVER TIME IS A MAIN GRADIENT OF HSP90 INHIBITION
INDUCED HIERARCHICAL
DESTABILIZATION 96
4.3.12 PROTEINS WITH PROTEIN KINASE ACTIVITY HAVE SHORTER HALF-LIFE 99
4.3.13 IDENTIFICATION OF HSP90 PHYSICAL INTERACTIONS BY IMMUNE
PRECIPITATION AND AFFINITY
PURIFICATION 100
4.3.14 DDR1 IS A NOVEL BONA FIDE CLIENT OF HSP90 102
4.4 CONCLUSION 104
SUMMARY 105
ZUSAMMENFASSUNG 107
REFERENCE 109
ABBREVIATIONS 124
APPENDIX 127
ACKNOWLEDGMENTS 135
CURRICULUM VITAE 136
I V
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| discipline | Biologie Medizin |
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| spelling | Wu, Zhixiang Verfasser aut Quantitative chemical proteomics for cancer characterization Zhixiang Wu 2012 IV, 137 S. Ill., graph. Darst. txt rdacontent n rdamedia nc rdacarrier Zsfassung in dt. Sprache München, Techn. Univ., Diss., 2012 Genanalyse (DE-588)4200230-8 gnd rswk-swf Proteinkinasen (DE-588)4175996-5 gnd rswk-swf Hitzeschock-Proteine (DE-588)4255487-1 gnd rswk-swf Krebs Medizin (DE-588)4073781-0 gnd rswk-swf (DE-588)4113937-9 Hochschulschrift gnd-content Krebs Medizin (DE-588)4073781-0 s Proteinkinasen (DE-588)4175996-5 s Genanalyse (DE-588)4200230-8 s Hitzeschock-Proteine (DE-588)4255487-1 s b DE-604 Erscheint auch als Online-Ausgabe urn:nbn:de:bvb:91-diss-20120511-1097813-1-3 http://mediatum.ub.tum.de/node?id=1097813 Verlag kostenfrei Volltext https://nbn-resolving.org/urn:nbn:de:bvb:91-diss-20120511-1097813-1-3 Resolving-System DNB Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=025135274&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Wu, Zhixiang Quantitative chemical proteomics for cancer characterization Genanalyse (DE-588)4200230-8 gnd Proteinkinasen (DE-588)4175996-5 gnd Hitzeschock-Proteine (DE-588)4255487-1 gnd Krebs Medizin (DE-588)4073781-0 gnd |
| subject_GND | (DE-588)4200230-8 (DE-588)4175996-5 (DE-588)4255487-1 (DE-588)4073781-0 (DE-588)4113937-9 |
| title | Quantitative chemical proteomics for cancer characterization |
| title_auth | Quantitative chemical proteomics for cancer characterization |
| title_exact_search | Quantitative chemical proteomics for cancer characterization |
| title_full | Quantitative chemical proteomics for cancer characterization Zhixiang Wu |
| title_fullStr | Quantitative chemical proteomics for cancer characterization Zhixiang Wu |
| title_full_unstemmed | Quantitative chemical proteomics for cancer characterization Zhixiang Wu |
| title_short | Quantitative chemical proteomics for cancer characterization |
| title_sort | quantitative chemical proteomics for cancer characterization |
| topic | Genanalyse (DE-588)4200230-8 gnd Proteinkinasen (DE-588)4175996-5 gnd Hitzeschock-Proteine (DE-588)4255487-1 gnd Krebs Medizin (DE-588)4073781-0 gnd |
| topic_facet | Genanalyse Proteinkinasen Hitzeschock-Proteine Krebs Medizin Hochschulschrift |
| url | http://mediatum.ub.tum.de/node?id=1097813 https://nbn-resolving.org/urn:nbn:de:bvb:91-diss-20120511-1097813-1-3 http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=025135274&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
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