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Analysis of Protein-Protein Interaction Byin Vivo Quantitative Proteomics In Analysis of protein-protein interaction by in vivo quantitative proteomics in Caenorhabditis elegans D i s s e r t a t i o n zur Erlangung des akademischen Grades d o c t o r r e r u m n a t u r a l i u m (Dr. rer. nat.) im Fach Biologie eingereicht an der Lebenswissenschaftlichen Fakultät der Humboldt-Universität zu Berlin von M.Sc. Jiaxuan Chen Präsident der Humboldt-Universität zu Berlin Prof. Dr. Jan-Hendrik Olbertz Dekan der Lebenswissenschaftlichen Fakultät Prof. Dr. Richard Lucius Gutachter/innen: 1. Prof. Dr. Richard Lucius 2. Prof. Dr. Matthias Selbach 3. Dr. Markus Landthaler Tag der mündlichen Prüfung: 21.09.2015 for my parents “No man is an island, entire of itself.” John Donne Table of Contents Table of Contents ................................................................................................................. I Abstract ............................................................................................................................... V Zusammenfassung ............................................................................................................ VI I Introduction ................................................................................................................ 7 I.1. Protein-protein interaction ........................................................................................................ 7 I.1.1. Detection of protein-protein interactions ................................................................................. 8 I.1.2. Affinity purification and mass spectrometry ............................................................................. 9 I.1.2.1 Tandem affinity purification ............................................................................................ 10 I.1.2.2 Affinity purification and quantitative proteomics ........................................................... 11 I.1.3. Negative control strategies ...................................................................................................... 12 I.1.4. In vivo protein-protein interactions ......................................................................................... 13 I.2. P granule biology in a nutshell ................................................................................................. 14 I.2.1. Germline development during early embryogenesis .............................................................. 14 I.2.2. P granule segregation during oocyte-to-embryo transition .................................................... 16 I.2.3. P granule composition and function ........................................................................................ 17 I.2.4. Regulation of P granule dynamics during early embryogenesis .............................................. 19 I.2.5. Other key players of anterior P granule disassembly? ............................................................ 20 I.3. Regulation of ribonulceoprotein granule assembly and disassembly in other systems .............. 22 I.3.1. Biophysical properties of proteins that promote liquid-liquid demixing ................................ 22 I.3.2. Regulation of RNP granule disassembly by kinase activity ...................................................... 23 I.4. Objectives of the thesis ............................................................................................................ 24 II Materials and Methods ........................................................................................... 26 II.1. Chemicals ................................................................................................................................. 26 II.2. Buffers and solutions ............................................................................................................... 26 II.2.1. Microbiology culture ................................................................................................................ 26 II.2.2. DNA sample preparation and electrophoresis ........................................................................ 26 II.2.3. SDS-PAGE and Western blotting .............................................................................................. 27 II.2.4. LC-MS sample preparation ...................................................................................................... 27 I | Page Table of Contents II.2.5. C. elegans culture and maintenance ....................................................................................... 28 II.2.6. C. elegans RNA interference .................................................................................................... 28 II.2.7. C. elegans embryo immunostaining ........................................................................................ 28 II.3. Kits and consumables ...............................................................................................................29 II.4. C. elegans cultivation ...............................................................................................................30 II.4.1. C. elegans strains ..................................................................................................................... 30 II.4.2. C. elegans culture ..................................................................................................................... 30 II.4.3. SILAC worm culture .................................................................................................................. 31 II.5. Mammalian standard and SILAC cell culture .............................................................................31 II.6. Cloning .....................................................................................................................................31 II.6.1. PCR ........................................................................................................................................... 31 II.6.2. Gateway recombination reaction ............................................................................................ 32 II.6.3. Transformation and plasmid DNA purification ........................................................................ 32 II.6.4. DNA sequencing ....................................................................................................................... 33 II.6.5. Construct generation ............................................................................................................... 33 II.7. Cell biology work ......................................................................................................................34 II.7.1. RNAi and sterility assays .......................................................................................................... 34 II.7.2. Mammalian cell transfection ................................................................................................... 35 II.7.3. Embryo immunostaining and mammalian cell fixation ........................................................... 35 II.7.4. Fluorescence microscopy ......................................................................................................... 36 II.8. Quantitative proteomics and affinity purification assays ..........................................................36 II.8.1. Benchmark for label-free quantification of whole proteomes ................................................ 36 II.8.2. Simulation of label-free pull-down experiments ..................................................................... 36 II.8.3. Embryo pull-down assays ........................................................................................................ 37 II.8.4. Pull-downs using SILAC worms to assess post-lysis bindings .................................................. 37 II.8.5. GEI-12 pull-downs in mammalian cells .................................................................................... 38 II.9. Ethanol protein precipitation and in-solution digestion ............................................................38 II.10. StageTip purification ................................................................................................................39 II.11. Liquid chromatography tandem mass spectrometry ................................................................. 39 II.12. Mass spectrometry data analysis ..............................................................................................40 II.13. SDS-PAGE and Western blotting ...............................................................................................42 II.14. Statistical and network analyses ..............................................................................................42 II | Page Table of Contents III Results ....................................................................................................................... 44 III.1. In vivo interaction proteomics method development ............................................................... 44 III.1.1. Technical assessment and optimization of label-free quantification ...................................... 44 III.1.1.1 Benchmark of whole proteome label-free quantification ............................................... 44 III.1.1.2 Simulation of label-free pull-down assays ....................................................................... 46 III.1.2. Optimization of specificity threshold ....................................................................................... 47 III.1.2.1 Definition of the combined
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