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Proteome and Metabolome Changes Associated with Mitochondrial Diseases

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Proteome and Metabolome Changes Associated with Mitochondrial Diseases Proteome and metabolome changes associated with mitochondrial diseases Inaugural-Dissertation to obtain the academic degree Doctor rerum naturalium (Dr. rer. nat.) submitted to the Department of Biology, Chemistry and Pharmacy of Freie Universität Berlin by Ina Aretz (nee Gielisch) from Quedlinburg Berlin, 2016 ii The present work was carried out from April 2012 until February 2016 at the Max Planck Institute for Molecular Genetics under the supervision of Dr. David Meierhofer. 1st Reviewer: Prof. Dr. Hans Lehrach 2nd Reviewer: Prof. Dr. Christian Freund Date of Defence: 07.06.2016 iii Für Zwicki. iv v Acknowledgements I would like to thank my PhD advisor Dr. David Meierhofer for giving me the oppor- tunity to work in his lab and on this exciting project. David provided me with guidance and support, but also gave me the freedom to develop and follow own research ideas. I also want to thank Prof. Dr. Hans Lehrach and Prof. Dr. Christian Freund for review- ing this PhD thesis. Many thanks go to my colleagues in the lab: Beata Lukaszewska-McGreal and Yang Ni, you guys provided such a friendly and helpful working environment that enabled me to finish this PhD work. Also, this work would never have been possible without the help of my co-authors from the Max Planck Institute and in Frankfurt. Therefore I would like to thank Christopher Hardt and Ilka Wittig. I also want to thank Jonas and Robin for proofreading this thesis and your useful com- ments. Besonders dankbar bin ich meinen Eltern, für die dauerhaft Unterstützung und Ermu- tigung, ohne die meine schulische und universitäre Ausbildung nicht möglich gewesen wäre. Finally, I would like to endlessly thank my love and best friend Jonas! vi Contents vii Contents Acknowledgements ...................................................................................................... v Contents ..................................................................................................................... vii Abstract ....................................................................................................................... xi Zusammenfassung .................................................................................................... xiii 1 Introduction ..................................................................................................... 1 1.1 Mitochondria and disease .............................................................................. 1 1.1.1 Mitochondrial structure ............................................................................. 1 1.1.2 Mitochondrial genetics .............................................................................. 2 1.1.3 The tricarboxylic acid cycle ...................................................................... 5 1.1.4 The electron transport chain and oxidative phosphorylation..................... 6 1.2 Approaches to mimic mitochondrial diseases ............................................... 9 1.3 Posttranslational modifications ................................................................... 10 1.4 Mass spectrometry ....................................................................................... 11 1.5 Mass spectrometry based proteomics .......................................................... 13 1.6 Mass spectrometry based metabolomics ..................................................... 16 1.6.1 Metabolomic approaches ......................................................................... 16 1.7 Omics technologies as a key to understand systems biology ...................... 19 1.8 Aim of the thesis ......................................................................................... 21 2 Material and Methods .................................................................................. 23 2.1 List of buffers .............................................................................................. 23 2.2 Cell culture .................................................................................................. 26 2.2.1 Rotenone treated HeLa cells .................................................................... 26 2.2.2 143B.TK- and ρ0 cells .............................................................................. 26 2.3 Verification of rotenone induced complex I deficiency in HeLa cells ....... 27 2.4 Verification of the ρ0 status by PCR ........................................................... 27 2.5 Cell harvesting and sample preparation for proteomics .............................. 28 2.5.1 Rotenone treated HeLa cells .................................................................... 28 2.5.2 143B.TK- and ρ0 cells .............................................................................. 28 2.6 Sample preparation for metabolomics ......................................................... 29 viii 2.6.1 Cell harvesting for metabolomics ............................................................ 29 2.6.2 Metabolite extraction ............................................................................... 29 2.7 Cell counting ................................................................................................ 30 2.8 Bicinchoninic acid assay .............................................................................. 31 2.9 DNA extraction ............................................................................................ 31 2.10 Gel electrophoresis ...................................................................................... 31 2.11 Sample clean-up ........................................................................................... 31 2.11.1 C18 cartridges .......................................................................................... 31 2.11.2 C18 stage tips ........................................................................................... 32 2.12 Ion exchange chromatography for sample fractionation ............................. 32 2.12.1 Strong anion exchange (SAX)-based fractionation of peptides ............... 32 2.12.2 Strong cation exchange (SCX) chromatography ..................................... 33 2.13 Analysis of posttranslational modifications (PTMs) by LC-MS ................. 34 2.13.1 Ubiquitylation .......................................................................................... 34 2.13.2 Phosphorylation ....................................................................................... 35 2.14 TCA cycle and OXPHOS enzyme activity measurements .......................... 35 2.14.1 Sample preparation for enzyme activity measurements .......................... 35 2.14.2 Fumarate dehydrogenase and malate dehydrogenase .............................. 36 2.14.3 Citrate synthase ........................................................................................ 36 2.14.4 Isocitrate dehydrogenase .......................................................................... 37 2.14.5 NADH dehydrogenase ............................................................................. 37 2.15 Metabolite tuning and LC-MS/MS optimization for MRM ion ratios ........ 38 2.15.1 Evaluation of MRMs and MRM ion ratios by amino acid calibration curves .............................................................................................................. 39 2.16 Metabolite profiling by targeted LC-MS/MS .............................................. 40 2.16.1 LC conditions for metabolomics .............................................................. 41 2.17 LC-MS settings for proteomics ................................................................... 42 2.18 Data analysis ................................................................................................ 43 2.18.1 Proteomics ................................................................................................ 43 2.18.2 Label free quantification - Rotenone treated HeLa cells ......................... 43 2.18.3 SILAC-based quantification - 143B.TK- and ρ0 cells .............................. 44 2.18.4 Metabolomics ........................................................................................... 47 3 Results ............................................................................................................. 49 Contents ix 3.1 Evaluation of MRMs and MRM ion ratios by amino acid calibration curves ..................................................................................................................... 50 3.1.1 Identifying inosine 5’-monophosphate by applying MRM ion ratios ..... 53 3.2 Rotenone induced complex I deficiency in HeLa cells ............................... 55 3.2.1 Verification of rotenone induced complex I deficiency in HeLa cells .... 55 3.2.2 Metabolome profiling of rotenone treated HeLa cells............................. 56 3.2.3 Proteome profiling of rotenone treated HeLa cells ................................. 58 3.3 Human cells lacking mtDNA ...................................................................... 70 3.3.1 Verification of the mtDNA depletion in ρ0 cells ..................................... 70 3.3.2 Metabolome profiling of ρ0 cells ............................................................. 71 3.3.3 Proteome profiling of ρ0 cells .................................................................. 73 3.3.4 Measurement of TCA cycle enzyme activities of ρ0 cells ....................... 86 4 Discussion ....................................................................................................... 87 4.1 Evaluation of
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