Availability: A systems biology driven approach for analyzing lipid protein interactions in sterol biosynthesis mutants

A systems biology driven approach for analyzing lipid protein interactions in sterol biosynthesis mutants:

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Bibliographic Details
Main Author:	Zauber, Henrik (Author)
Format:	Thesis/Dissertation Book
Language:	English
Published:	2013
Subjects:	Biosynthese Sterine Protein-Protein-Wechselwirkung Hochschulschrift
Links:	http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=026873106&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA
Physical Description:	4, 126 Bl. Ill., graph. Darst. CD-ROM (12 cm)

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adam_text	CONTENTS ABBREVIATIONS 4 1 GENERAL INTRODUCTION 7 1.1 STEROL-BIOSYNTHESIS MUTANTS 8 1.1.1 THE SMTL STEROL-BIOSYNTHESIS MUTANT 8 1.1.2 THE UGT80A2;BL STEROL-BIOSYNTHESIS MUTANT 9 1.2 STRUCTURAL LIPIDS IN THE PLASMA MEMBRANE 9 1.2.1 STRUCTURAL GLYCEROPHOSPHOLIPIDS 9 1.2.2 PHYTOSTEROLS 10 1.2.3 SPHINGOLIPIDS 10 1.3 STRATEGIES IN PROTEOMIC DATA ANALYSES 11 1.4 AIMS OF THE WORK 12 2 AUTOMATED STANDARDIZED DATA ANALYSIS OF LC/MS DERIVED PROTEOMIC DATA 13 2.1 INTRODUCTION 15 2.2 RESULTS 17 2.2.1 INSTALLATION OF CRACKER 17 2.2.2 IMPORT OF PEPTIDE LISTS 17 2.2.3 GENERAL PROCESSING OF PEPTIDE INTENSITY LISTS 17 2.2.4 USE OF EMPAI AS A RELATIVE PROTEIN ABUNDANCE MEASURE WITHIN SAMPLES .... 18 2.2.5 CRACKER INCLUDES MULTIVARIATE STATISTICAL ANALYSIS 19 2.2.6 EXPERIMENTAL DESIGN 19 2.2.7 ANALYSIS OF PHOSPHOPEPTIDE ENRICHED SAMPLES 19 2.2.8 EXAMPLE OF CRACKER FUNCTIONS 20 2.2.9 VALIDATION WITH EXISTING WORKFLOWS 23 2.3 DISCUSSION 23 2.3.1 CRACKER SPEEDS UP AND STANDARDIZES PROTEOMIC DATA MINING 23 2.3.2 CRACKER ANALYSIS REPRODUCES PUBLISHED KNOWLEDGE 24 2.4 METHODS 25 2.4.1 SAMPLE PREPARATION 25 2.4.2 PROTEOMIC ANALYSIS 25 2.4.3 SUPPLEMENTARY 26 HTTP://D-NB.INFO/1043807942 CONTENTS 3 SYSTEMATIC EVALUATION OF REFERENCE PROTEIN NORMALIZATION IN PROTEOMIC EXPERIMENTS 27 3.1 INTRODUCTION 29 3.2 RESULTS 30 3.2.1 RELATIONSHIP BETWEEN PEPTIDE INTENSITIES AND SPIKED BSA AMOUNTS 30 3.2.2 INFLUENCE OF DIFFERENT TOTAL PROTEIN BACKGROUND 30 3.2.3 VALIDATION OF RPN IN THE CONTEXT OF NUTRIENT STARVATION 33 3.2.4 VALIDATION OF RPN APPLICABILITY FOR ABSOLUTE QUANTITATION 33 3.3 DISCUSSION 35 3.3.1 BSA AS AN INTERNAL REFERENCE STANDARD 35 3.3.2 APPLICATION OF RPN IN COMPARATIVE PROTEOMICS 37 3.3.3 CONCLUSIONS 38 3.4 METHODS 39 3.4.1 CELL SUSPENSION CULTURING AND NUTRIENT DEPLETION EXPERIMENTS 39 3.4.2 SAMPLE PREPARATION 39 3.4.3 SAMPLE PREPARATION FOR PROTEOMIC ANALYSIS 39 3.4.4 LC-MS/MS ANALYSIS AND PROTEIN IDENTIFICATION 39 3.4.5 QUANTITATION AND REFERENCE PROTEIN NORMALIZATION 40 3.4.6 ABSOLUTE QUANTITATION USING RPN 40 3.4.7 STATISTICS 41 3.4.8 PROTOCOL FOR EVALUATING RPN 41 3.5 SUPPLEMENTARY 41 4 UNRAVELING STEROL-DEPENDENT MEMBRANE PHENOTYPES UNDER STEROL-DEPLETION 43 4.1 INTRODUCTION 45 4.2 RESULTS 46 4.2.1 REPRODUCIBILITY OF UNICORN RESULTS 46 4.2.2 SIGNIFICANT ALTERATIONS IN DRM/DSF RATIOS IN BOTH UPON M\|3CD TREATMENT AND SMTL MUTANT 49 4.2.3 EFFICIENT REMOVAL OF CO-PURIFYING PROTEINS BY PAIRWISE ANALYSIS OF FRACTIONS . 51 4.2.4 STEROL-DEPENDENT PROTEINS WITH MAJOR FUNCTIONS IN SIGNALING AND TRANSPORT. . 51 4.2.5 DEPLETION OF MICRODOMAIN RELATED PROTEINS FROM DRM AFTER STEROL DEPLETION . 53 4.3 DISCUSSION 53 4.3.1 BOOTSTRAPPING AS A SUITABLE SCORING METHOD 55 4.3.2 EFFICIENT CHARACTERIZATION OF STEROL-DEPENDENT PROTEINS BY ABUNDANCE DISTRI BUTIONS 55 4.4 CONCLUSION 58 4.5 METHODS 58 4.5.1 CELL SUSPENSION CULTURE 58 4.5.2 SAMPLE PREPARATION AND PROTEIN EXTRACTION 59 4.5.3 PROTEOMIC ANALYSIS 59 4.5.4 LC-MS/MS ANALYSIS AND PROTEIN IDENTIFICATION 60 4.5.5 CALCULATION OF LABEL-FREE PROTEIN INTENSITIES 60 4.5.6 BOOTSTRAP APPROACH FOR ANALYSIS OF DRM/DSF ABUNDANCE RATIOS 60 4.5.7 STATISTICAL ANALYSIS OF STEROL-DEPENDENT PROTEIN CANDIDATES 61 4.6 SUPPLEMENTARY 61 CONTENTS 5 A SYSTEMS BIOLOGY DRIVEN APPROACH FOR ANALYZING LIPID PROTEIN INTERACTIONS 63 5.1 INTRODUCTION 65 5.2 RESULTS 66 5.2.1 DETECTION OF STEROL-DEPENDENT PROTEIN CANDIDATES BY DRM/DSF DISTRIBUTION ANALYSIS 66 5.2.2 ALTERATION IN LIPID PROFILES IN BOTH MUTANTS 68 5.2.3 CORRELATING ALTERATIONS BETWEEN LIPID SYNTHESIS-RELATED PROTEINS AND LIPID ABUNDANCE PROFILES 70 5.2.4 THE LINK BETWEEN STEROL-DEPENDENT PROTEINS AND CERAMIDES 70 5.3 DISCUSSION 75 5.3.1 RELATIONSHIP BETWEEN DEPLETION OF DRM-RELEVANT MICRODOMAIN PROTEINS AND STRUCTURAL CHANGES IN MICRODOMAINS 77 5.3.2 EVIDENCE FOR STEROLS AS DONORS OF GLYCOSYLRESIDUES IN A CERAMIDE GLYCOSYLA- TION PATHWAY 78 5.3.3 INVERSE EFFECTS ON LIPID SYNTHESIS PATHWAYS BETWEEN BOTH MUTANTS 79 5.3.4 ALTERATIONS IN SIGNALING PATHWAYS LOCALIZED AT THE PLASMA MEMBRANE 79 5.3.5 GENERAL IMPLICATIONS OF LIPID-PROTEIN INTERACTIONS IN PLASMA MEMBRANE FUNC TIONS 82 5.4 CONCLUSION 83 5.5 METHODS 84 5.5.1 CELL SUSPENSION CULTURING 84 5.5.2 SAMPLE PREPARATION AND PROTEIN EXTRACTION 84 5.5.3 LIPID EXTRACTION 84 5.5.4 LC-MS/MS ANALYSIS AND PROTEIN IDENTIFICATION 85 5.5.5 PEAK IDENTIFICATION AND QUANTIFICATION OF LIPIDS 85 5.5.6 STATISTICAL ANALYSIS 85 5.5.7 NETWORK ANALYSIS 86 5.6 SUPPLEMENTARY 86 6 GENERAL DISCUSSION 88 6.1 SYSTEMS APPROACHES NEED AUTOMATION OF DATA ANALYSIS 88 6.2 CALLUS AS A USEFUL SYSTEM FOR STUDYING STEROL BIOSYNTHESIS MUTANTS 89 6.3 LABEL-FREE PROTEOMICS ARE SUFFICIENT FOR ANALYSIS OF STEROL-DEPENDENT PROTEINS 89 6.4 COMPARATIVE ANALYSIS OF UGT80A2;BL AND SMTL MUTANT REVEALED CONTRARY CELL STATUS BETWEEN MUTANTS 91 6.5 STRUCTURAL ALTERATIONS IN PLASMA MEMBRANE ARE OVERLAID BY SIGNALING PROCESSES ... 91 6.6 CRITICISM ON USAGE OF DETERGENTS IN MICRODOMAIN RESEARCH 92 6.7 KEY RESULTS 93 SUMMARY 94 BIBLIOGRAPHY 95 SUPPLEMENTAL INFORMATIONS 116 ACKNOWLEDGEMENTS 128 EHRENWDRTLICHE ERKLARUNG 129 CONTRIBUTIONS OF THE AUTHORS 130
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spellingShingle	Zauber, Henrik A systems biology driven approach for analyzing lipid protein interactions in sterol biosynthesis mutants Biosynthese (DE-588)4006902-3 gnd Sterine (DE-588)4126502-6 gnd Protein-Protein-Wechselwirkung (DE-588)4745193-2 gnd
subject_GND	(DE-588)4006902-3 (DE-588)4126502-6 (DE-588)4745193-2 (DE-588)4113937-9
title	A systems biology driven approach for analyzing lipid protein interactions in sterol biosynthesis mutants
title_auth	A systems biology driven approach for analyzing lipid protein interactions in sterol biosynthesis mutants
title_exact_search	A systems biology driven approach for analyzing lipid protein interactions in sterol biosynthesis mutants
title_full	A systems biology driven approach for analyzing lipid protein interactions in sterol biosynthesis mutants Henrik Zauber
title_fullStr	A systems biology driven approach for analyzing lipid protein interactions in sterol biosynthesis mutants Henrik Zauber
title_full_unstemmed	A systems biology driven approach for analyzing lipid protein interactions in sterol biosynthesis mutants Henrik Zauber
title_short	A systems biology driven approach for analyzing lipid protein interactions in sterol biosynthesis mutants
title_sort	a systems biology driven approach for analyzing lipid protein interactions in sterol biosynthesis mutants
topic	Biosynthese (DE-588)4006902-3 gnd Sterine (DE-588)4126502-6 gnd Protein-Protein-Wechselwirkung (DE-588)4745193-2 gnd
topic_facet	Biosynthese Sterine Protein-Protein-Wechselwirkung Hochschulschrift
url	http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=026873106&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA
work_keys_str_mv	AT zauberhenrik asystemsbiologydrivenapproachforanalyzinglipidproteininteractionsinsterolbiosynthesismutants

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