Development of new approaches for kinase-centric proteomics [Elektronische Ressource] / Felix Sebastian Oppermann

Max Planck Institute Of Biochemistry

Sujets

• Biologie

Informations

• Publié par: technische_universitat_munchen
• Publié le: 01 janvier 2010
• Nombre de lectures: 40
• Langue: English
• Poids de l'ouvrage: 6 Mo

Extrait

Technische Universität München
Max-Planck-Institut für Biochemie


Development of new approaches for kinase-centric
proteomics



Felix Sebastian Oppermann



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 23.08.2010 bei der Technischen Universität München
eingereicht und durch die Fakultät für Chemie am 01.12.2010 angenommen.











for my parents











Content

I. Introduction ........................................................................................................... 1
1.1 Protein kinases in health and disease .............................. 1
1.1.1 Protein kinases and phosphorylation-based signaling ............................. 1
1.1.2 Control of substrate specificity ............................................................... 2
1.1.3 Protein kinases and human cancer .......................... 3
1.1.4 Inhibiting protein kinase activity ............................. 4
1.2 Chronic myeloid leukemia and Bcr-Abl ................................ 5
1.2.1 Chronic myeloid leukemia ........................................... 5
1.2.2 Protein domains of Bcr-Abl and leukemogenic signaling ........................... 7
1.2.3 Therapeutic strategies for treating chronic myeloid leukemia ..................... 9
2.4 Imatinib and resistance formation ................................................................. 10
1.3 Polo-like kinase 1, a mitotic Serine/Threonine kinase ...... 12
1.3.1 Structural properties and activity control of Polo-like kinase 1 ................. 12
1.3.2 Mitotic functions of Polo-like kinase 1 ...................................................... 13
1.3.3 Polo like kinase 1 and human cancer ......................... 16
1.4 Mass spectrometry-based proteomics ............................................................... 17
1.4.1 Mass spectrometry applied to protein research .......................................... 17
1.4.2 Quantitative proteomics ............................................. 19
1.4.3 Phosphoproteomics .................................................... 21
1.5 Characterizing protein kinase inhibitors and kinase signaling .......................... 22
1.5.1 Chemical proteomics .................................................. 22
1.5.2 Chemical genetics ...................................................... 24
II. Aims of the thesis ................................................................... 26
III. Materials and Methods .......................... 28
3.1 Material sources ................................ 28
3.1.1 Laboratory chemicals and biochemicals .................... 28
3.1.2 Chemicals for SILAC and MS-analysis ..................................................... 28
3.1.3 Other materials ........................................................... 29
3.2 Cell culture Media ............................. 29
3.3 Stock solutions and commonly used buffers ..................................................... 29
3.4 Cells ................................................................................... 30
3.5 Antibodies ......................................... 31
3.5.1 Primary antibodies ...................... 31
3.5.2 Secondary antibodies .................................................................................. 31
3.6 Cell culture .................................... 32
3.7 Protein analytical methods ................ 32
3.7.1 Determination of protein concentration in cell lysates ............................... 32
3.7.2 SDS-polyacrylamide-gelelectrophoresis (SDS-PAGE) ............................. 32
3.7.3 Transfer of proteins onto nitrocellulose membranes .................................. 32
3.7.4 Immunoblot detection ................................................ 32
3.8 SILAC labeling, cell lysis and anti-pTyr immunoprecipitation ........................ 33
3.8.1 Cell culture in SILAC medium .................................................................. 33
3.8.2 Cell lysis with Tritron X-100 ..... 34
3.8.3 Cell lysis with NP-40 and anti-pTyr immunoprecipitation ........................ 34
3.8.4 Cell lysis with 8M Urea ............................................................................. 35
3.9 Generation of kinase inhibitor resins and kinase-affinity enrichment .............. 35
3.9.1 Study for the quantitative comparison of kinase inhibitor resins ............... 35
3.9. Study for the quantitative comparison of relative kinase expression levels 36
3.9.3 Study for the qualitative comparison of phosho-kinomes .......................... 36
3.9.4 Study for the identification of cellular imatinib targets and imatinib-
sensitive phosphorylation sites ............................................................................ 36
3.10 Mass spectrometry sample preparation ........................................................... 37
3.10.1 In-solution protein digest ......... 37
3.10.2 In-gel protein digest ................................................................................. 38
3.10.3 Titanium dioxide microsphere-based enrichment of phosphopeptides .... 38
3.10.4 Immobilized metal affinity chromatography for phosphopeptide
enrichment ........................................................................................................... 39
3.10.5 Strong cation exchange chromatography for phosphopeptide separation
(ResourceS column) ............................................................................................ 39
3.10.5 Strong cation exchange chromatography for phosphopeptide separation
(polySULFOETHYL A column) ........................................................................ 39
3.10.6 Sample processing for the comparison of inhibitor-resins, kinase
expression levels and the phospho-kinome analysis ........... 40
3.10.7 Sample processing for the identification of imatinib targets and imatinib-
sensitive phosphorylation events ......................................................................... 40
3.10.8 Sample processing for the cellular substrate identification for Polo-like-
kinase 1 ................................................ 40
3.11 MS analysis and data processing ..................................................................... 41
3.11.1 MS analysis on the LTQ-Orbitrap ............................ 41
3.11.2 Peptide identification, quantification and data analysis (MSQuant) ........ 41
3.11.3 Peptide identification and quantification (MaxQuant) ............................. 43
3.11.4 Determine Ratio similarity coefficient curves (RSC) .............................. 43
3.11.5 Gene ontology analysis ............................................................................ 44
3.11.6 Phosphorylation site overlap between technical and biological replicates.
............................................................................................................................. 44
IV. Results ................... 45
4.1 Large-scale Proteomics Analysis of the Human Kinome 46
4.1.1 Comparative profiling for kinase-selective pre-fractionation reagents ...... 46
4.1.2 Comparative Kinase Expression Analysis in Different Cancer Cell Lines 54
4.1.3 Benchmark analysis of the PTM scoring algorithm ................................... 59
4.1.4 Kinase-centric phosphoproteomics analysis of cancer cell lines ............. 61
4.2 Identification of cellular imatinib targets and imatinib-sensitive phosphorylation
sites .......................................................................................................................... 69
4.2.1 Multicolumn-based protein kinase affinity chromatography ..................... 69
4.2.2 Parallel, batch-wise processing for protein kinase enrichment .................. 73
4.2.3 Analysis of the phosphotyrosine-containing sub-proteome upon imatinib
treatment .............................................................................................................. 80
4.3 Identification of Plk1 cellular substrates ...................... 84
4.3.1 Implementation of an efficient phosphopeptide enrichment strategy ........ 84
4.3.2 New strategy for the identification of cellular substrates of Plk1 .............. 86
4.3.3 Characterization of the identified cellular substrates of Plk1 .................... 91
V. Discussion .............................................................................................................. 94
5.1. Large-scale proteomics analysis of the human kinome ... 94
5.1.1 Pyrido[2,3-d]pyrimidin-based affinity resins are efficient protein kinase
pre-fractionation tools ......................................................................................... 94
5.1.2 Comparative analysis of kinase expression in three cancer cell lines ........ 96
5.1.3 Be