Quantitative proteomics strategies to reveal cellular mechanisms of kinase inhibitor drug action:
Gespeichert in:
| 1. Verfasser: | |
|---|---|
| Format: | Abschlussarbeit Buch |
| Sprache: | English |
| Veröffentlicht: |
2013
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| Schlagworte: | |
| Online-Zugang: | Volltext https://nbn-resolving.org/urn:nbn:de:bvb:91-diss-20130923-1144908-0-7 Inhaltsverzeichnis |
| Beschreibung: | 159 S. graph. Darst. |
Internformat
MARC
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| 100 | 1 | |a Weber, Christoph Alfred Willibald |e Verfasser |4 aut | |
| 245 | 1 | 0 | |a Quantitative proteomics strategies to reveal cellular mechanisms of kinase inhibitor drug action |c Christoph Alfred Willibald Weber |
| 264 | 1 | |c 2013 | |
| 300 | |a 159 S. |b graph. Darst. | ||
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| 337 | |b n |2 rdamedia | ||
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| 502 | |a München, Techn. Univ., Diss., 2013 | ||
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| 912 | |a ebook | ||
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Datensatz im Suchindex
| _version_ | 1804151469801209856 |
|---|---|
| adam_text | TABLE OF CONTENTS
I INTRODUCTION 1
1. CELLULAR SIGNAL TRANSDUCTION VIA POSTTRANSLATIONAL MODIFICATIONS 1
2. PROTEIN PHOSPHORYLATION 5
2.1 APPLICABILITY OF PHOSPHORYLATION IN A BIOLOGICAL SYSTEM 6
2.2 PROTEIN KINASE CLASSIFICATION STRUCTURAL PROPERTIES 6
3. PROTEIN KINASES AND CANCER 10
3.1 THE CONNECTION BETWEEN KINASES AND MALIGNANT PHENOTYPES 10
3.2 PROTEIN KINASE INHIBITION 11
4. MS-BASED PROTEOMICS 15
4.1 PROTEOMICS 15
4.2 MASS SPECTROMETRY ANALYSIS OF PROTEINS 15
4.3 FUNDAMENTALS OF QUANTITATIVE MASS SPECTROMETRY 21
4.4 AFFINITY CHROMATOGRAPHY-BASED PROTEOMICS 24
4.4.1 PRINCIPLES OF CHEMICAL PROTEOMICS 25
4.4.2 CHEMICAL PROTEOMICS FOR CHARACTERIZING KINASE INHIBITORS 26
4.4.3 PROTEOMIC EXAMINATION OF PROTEIN-PROTEIN INTERACTIONS 27
4.5 PHOSPHOPROTEOMICS 29
4.5.1 ENRICHMENT OF PHOSPHORYLATED PEPTIDES 31
4.5.2 PHOSPHOPEPTIDE FRACTIONATION 32
4.5.3 QUANTITATIVE MASS SPECTROMETRY ANALYSIS OF PHOSPHORYLATED PEPTIDES
32
5. ACUTE MYELOID LEUKEMIA 34
II MATERIALS AND METHODS 37
1. MATERIALS 37
1.1 LABORATORY CHEMICALS AND BIOCHEMICALS 37
1.2 SMALL MOLECULES 38
1.3 BIOLOGICALS 38
1.3.1 LIGANDS 38
1.3.2 ANTIBODIES 39
1.3.3 PEPTIDES 39
HTTP://D-NB.INFO/104384080X
1.3.4 ENZYMES AND SUBSTRATES
1.3.5 COMPONENTS OF MOLECULAR WEIGHT STANDARD
1.4 CONSUMABLES
1.5 KITS
1.6 BUFFERS AND SOLUTIONS
1.6.1 COMMERCIAL
1.6.2 HOME-MADE
1.7 CELL CULTURE MEDIA AND SUPPLEMENTS
1.8 CELL LINES
1.9 INSTRUMENTS
1.10 SOFTWARE AND ONLINE TOOLS
2. METHODS
2.1 CELL CULTURE
2.1.1 STANDARD MAINTENANCE
2.1.2 SILAC LABELING
2.2 TREATMENT, CELL HARVEST AND LYSIS
2.2.1 LYSIS WITH
TRITON X-100 LYSIS BUFFER
2.2.2 LYSIS WITH UREA LYSIS BUFFER
2.3 IN VITRO ASSOCIATION EXPERIMENTS
2.4 SDS-POLYACRYLAMIDE-GELELECTROPHORESIS (SDS-PAGE)
2.5 WESTERN BLOTTING
2.6 SAMPLE PREPARATION FOR (PHOSPHOPROTEOMICS) ANALYSIS
2.6.1 IN-GEL DIGESTION
2.6.2 IN-SOLUTION DIGESTION
2.6.3 DESALTING OF PEPTIDE MIXTURES
2.6.3.1 C18 SEP-PAK CARTRIDGES
2.6.3.2 STAGETIPS
2.6.4 STRONG CATION EXCHANGE CHROMATOGRAPHY
2.6.5 IMMOBILIZED METAL AFFINITY CHROMATOGRAPHY ENRICHMENT OF
PHOSPHOPEPTIDES (IMAC)
2.7 KINASE ASSAYS
2.8 CELLULAR CDLLB STAINING FOR FACS ANALYSIS
2.9 CELL VIABILITY ASSAY
.39
.40
.40
.41
.41
.41
.41
.44
.45
.45
.46
.46
.46
.46
.47
.47
.47
.47
.48
.49
.49
.50
.50
.50
.51
.51
.51
,51
.52
.52
..53
..54
2.10 MASS SPECTROMETRIC ANALYSIS 54
2.11 PROCESSING OF MS DATA 55
2.11.1 MAXQUANT 55
2.11.2 DATA ANALYSIS 56
2.11.2.1 PHOSPHOPROTEOMICS 56
2.11.2.2 CHEMICAL PROTEOMICS 56
III RESULTS 59
1. PROTEOMICS STRATEGY FOR QUANTITATIVE PROTEIN INTERACTION PROFILING 59
1.1 CONCEPTUAL OUTLINE OF THE QUANTITATIVE IN VITRO ASSOCIATION STRATEGY
FOR TARGET AFFINITY
DETERMINATIONS 61
1.2 QUANTITATIVE CHEMICAL PROTEOMICS PROFILING OF GEFITINIB 63
1.2.1 AFFINITY CHROMATOGRAPHY WITH IMMOBILIZED AX14596 AS CAPTURING
MOLECULE 63
1.2.2 COMPETITION WITH FREE GEFITINIB 70
1.3 QUANTITATIVE CHEMICAL PROTEOMICS PROFILING OF SB203580 74
1.3.1 IN VITRO ASSOCIATION EXPERIMENTS WITH IMMOBILIZED VI16742 AS
CAPTURING MOLECULE 74
1.3.1.1 GENE ONTOLOGY ANALYSIS OF VI16742 BINDERS 77
1.3.2 COMPETITION WITH FREE SB203580 78
1.4 COMPARISON WITH ANOTHER QUANTITATIVE CHEMICAL PROTEOMICS TECHNIQUE
80
1.5 QUANTITATIVE PROFILING OF PEPTIDE-PROTEIN INTERACTIONS 83
1.6 PROFILING OF PROTEIN-PROTEIN INTERACTIONS 87
2. QUANTITATIVE PROTEOMIC ANALYSIS OF GEFITINIB S AND ERLOTINIB S
EFFECTS IN AMI CELLS 89
2.1 EXPERIMENTAL STRATEGY 90
2.2 QUALITATIVE PHOSPHOPROTEOMICS ANALYSIS OF KG-1 CELLS 92
2.2.1 GENE ONTOLOGY ANALYSIS 93
2.2.2 ANALYSIS OF PHOSPHORYLATIONS WITHIN THE ACTIVATION LOOPS OF
KINASES 94
2.2.3 EXTRACTION OF OVERREPRESENTED PHOSPHORYLATION MOTIFS 95
2.2.4 PHOSPHORYLATION OF HISTONE DEACETYLASES 96
2.3 QUANTITATIVE ANALYSIS OF GEFINTIB- AND ERLOTINIB-INDUCED
PHOSPHOREGULATION 98
2.4 QUANTITATIVE CHEMICAL PROTEOMIC PROFILING OF GEFITINIB AND ERLOTINIB
IN KG-1 CELLS 105
IV DISCUSSION 109
1. QUANTITATIVE PROTEIN INTERACTION PROFILING 109
1.1 METHODICAL REMARKS 109
1.1.1 QUANTITATIVE IN VITRO ASSOCIATION EXPERIMENTS 109
1.1.2 PROFILING OF SOLUBLE SMALL-MOLECULE KINASE INHIBITORS 112
1.2 CELLULAR TARGETS OF GEFITINIB 114
1.3 CELLULAR TARGETS OF SB203580 115
1.4 INTERACTION PARTNERS OF PHOSPHORYLATED IRS4 PEPTIDE 116
1.5 PROFILING OF EGFR INTERACTION PARTNERS 117
2. PHOSPHOPROTEOMICS AND CHEMICAL PROTEOMICS CHARACTERIZATION OF
ERLOTINIB AND GEFITINIB
INTERFERENCE IN AML CELLS 118
V SUMMARY 127
VI REFERENCES 129
VII APPENDIX 147
1. ABBREVIATIONS 147
2. ADDITIONAL TABLE .151
3. LIST OF FIGURES 157
4. LIST OF TABLES 158
5. PUBLICATIONS 158
6. ACKNOWLEDGMENTS 159
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| spelling | Weber, Christoph Alfred Willibald Verfasser aut Quantitative proteomics strategies to reveal cellular mechanisms of kinase inhibitor drug action Christoph Alfred Willibald Weber 2013 159 S. graph. Darst. txt rdacontent n rdamedia nc rdacarrier München, Techn. Univ., Diss., 2013 (DE-588)4113937-9 Hochschulschrift gnd-content Erscheint auch als Online-Ausgabe urn:nbn:de:bvb:91-diss-20130923-1144908-0-7 http://mediatum.ub.tum.de/node?id=1144908 Verlag kostenfrei Volltext https://nbn-resolving.org/urn:nbn:de:bvb:91-diss-20130923-1144908-0-7 Resolving-System DNB Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=026824594&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Weber, Christoph Alfred Willibald Quantitative proteomics strategies to reveal cellular mechanisms of kinase inhibitor drug action |
| subject_GND | (DE-588)4113937-9 |
| title | Quantitative proteomics strategies to reveal cellular mechanisms of kinase inhibitor drug action |
| title_auth | Quantitative proteomics strategies to reveal cellular mechanisms of kinase inhibitor drug action |
| title_exact_search | Quantitative proteomics strategies to reveal cellular mechanisms of kinase inhibitor drug action |
| title_full | Quantitative proteomics strategies to reveal cellular mechanisms of kinase inhibitor drug action Christoph Alfred Willibald Weber |
| title_fullStr | Quantitative proteomics strategies to reveal cellular mechanisms of kinase inhibitor drug action Christoph Alfred Willibald Weber |
| title_full_unstemmed | Quantitative proteomics strategies to reveal cellular mechanisms of kinase inhibitor drug action Christoph Alfred Willibald Weber |
| title_short | Quantitative proteomics strategies to reveal cellular mechanisms of kinase inhibitor drug action |
| title_sort | quantitative proteomics strategies to reveal cellular mechanisms of kinase inhibitor drug action |
| topic_facet | Hochschulschrift |
| url | http://mediatum.ub.tum.de/node?id=1144908 https://nbn-resolving.org/urn:nbn:de:bvb:91-diss-20130923-1144908-0-7 http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=026824594&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
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