TECHNISCHE UNIVERSITÄT MÜNCHEN Max-Planck-Institut für Biochemie Quantitative Proteomics Strategies to Reveal Cellular Mechanisms of Kinase Inhibitor Drug Action Christoph Alfred Willibald Weber Vollständiger Abdruck der von der Fakultät für Chemie der Technischen Universität München zur Erlangung des akademischen Grades eines Doktors der Naturwissenschaften genehmigten Dissertation. Vorsitzender: Univ.-Prof. Dr. Michael Groll Prüfer der Dissertation: 1. Priv.-Doz. Dr. Henrik Daub 2. Univ.-Prof. Dr. Johannes Buchner Die Dissertation wurde am 07.05.2013 bei der Technischen Universität München eingereicht und durch die Fakultät für Chemie am 19.08.2013 angenommen. Meinen Eltern 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 spectrometric 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 1.3.4 Enzymes and substrates .................................................................................................. 39 1.3.5 Components of molecular weight standard ................................................................... 40 1.4 Consumables ........................................................................................................................... 40 1.5 Kits ........................................................................................................................................... 41 1.6 Buffers and solutions .............................................................................................................. 41 1.6.1 Commercial ..................................................................................................................... 41 1.6.2 Home-made .................................................................................................................... 41 1.7 Cell culture media and supplements ...................................................................................... 44 1.8 Cell lines .................................................................................................................................. 45 1.9 Instruments ............................................................................................................................. 45 1.10 Software and online Tools ...................................................................................................... 46 2. Methods .......................................................................................................................................... 46 2.1 Cell culture .............................................................................................................................. 46 2.1.1 Standard Maintenance .................................................................................................... 46 2.1.2 SILAC labeling .................................................................................................................. 47 2.2 Treatment, cell harvest and lysis ............................................................................................ 47 2.2.1 Lysis with triton X-100 lysis buffer .................................................................................. 47 2.2.2 Lysis with urea lysis buffer .............................................................................................. 47 2.3 In vitro association experiments ............................................................................................. 48 2.4 SDS-polyacrylamide-gelelectrophoresis (SDS-PAGE) .............................................................. 49 2.5 Western Blotting ..................................................................................................................... 49 2.6 Sample preparation for (phosphoproteomics) analysis ......................................................... 50 2.6.1 In-gel digestion ................................................................................................................ 50 2.6.2 In-solution digestion ....................................................................................................... 50 2.6.3 Desalting of peptide mixtures ......................................................................................... 51 2.6.3.1 C18 Sep-Pak cartridges ................................................................................................ 51 2.6.3.2 StageTips ..................................................................................................................... 51 2.6.4 Strong cation exchange chromatography ....................................................................... 51 2.6.5 Immobilized metal affinity chromatography enrichment of phosphopeptides (IMAC) . 52 2.7 Kinase assays ........................................................................................................................... 52 2.8 Cellular CD11b staining for FACS analysis ............................................................................... 53 2.9 Cell viability assay ................................................................................................................... 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 ..............................................................................................