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Using Proteomics to Elucidate Critical Signaling Pathways Involved in Hematopoietic Differentiation and Migration Using Proteomics To Elucidate Critical Signaling Pathways Involved In Hematopoietic Differentiation And Migration Thesis by Heba Ahmed In partial fulfillment of the requirements For the degree of Master of science King Abdullah University of Science and Technology Thuwal, Kingdom of Saudi Arabia November 2012 2 EXAMINATION COMMITTEE APPROVALS FORM The thesis of Heba Ahmed is approved by the examination committee. Committee Chairperson: Jasmeen Merzeban Committee Member: Christopher Gehring Committee Member: Samah Zeineb Gadhoum 3 . © November 2012 Heba Ahmed All Rights Reserved 4 ABSTRACT Using Proteomics To Elucidate Critical Signaling Pathways Involved In Hematopoietic Differentiation And Migration Heba Ahmed Despite important advances in the therapy of acute myeloid leukemia (AML) the majority of patients will die from their disease (Appelbaum, Rowe, Radich, & Dick, 2001). Characterization of the aberrant molecular pathways responsible for this malignancy provides a platform to discover alternative treatments to help alter the fate of patients. AML is characterized by a blockage in the differentiation of myeloid cells resulting in the accumulation of highly proliferating immature hematopoietic cells. Since treatments such as chemotherapy rarely destroy the leukemic cells entirely, differentiation induction therapy has become a very attractive treatment option. Interestingly, previous experiments have shown that ligation of CD44, a cell surface glycoprotein strongly expressed on all AML cells, with anti-CD44 monoclonal antibodies (mAbs) could reverse this block in differentiation of leukemic blasts regardless of the AML subtype. To expand the understanding of the cellular regulation and 5 circuitry involved, we aim to apply quantitative phosphoproteomics to monitor dynamic changes in phosphorylation state in response to anti- CD44 treatment. Protein phosphorylation and dephosphorylation is a highly controlled biochemical process that responds to various intracellular and extracellular stimuli. As phosphorylation is a dynamic process, quantification of these phosphorylation events would be vastly insightful. The main objective of this project is to determine the differentiation- dependent phosphoproteome of AML cells upon treatment of cells with the anti-CD44 mAb. In these experiments, optimization of protein extraction, phosphopeptide enrichment and data processing and analysis has been achieved. The primary results show successful phosphoproteome extraction complemented with efficient phosphopeptide enrichment and informative data processing. Further quantification with stable isotope labeling techniques is anticipated to provide candidates for targeted therapy. 6 ACKNOWLEDGEMENTS Foremost, I would like to express my sincere gratitude to my Supervisor Professor: Jasmeen Merzaban for the continuous support of my study and research, for her patience, motivation, enthusiasm, and immense knowledge. And thanks to Dr. Zeineb Gadhoum and Dr. Kosuke Sakashita for their help and guidance throughout the time of my research. I would also like to thank my committee member Professor Chrisopher Gehring for his guidance and support during my Masters education. I thank my fellow lab mates, Dina Abu Samra, Nour Madhoun, Amal Ali and Ayman El Khodiery for bearing with all my endless questions interrupting their lab work, for our stimulating discussions, and for all the fun we have had. Thanks to Maryame Mih for her great efforts in managing the lab and providing the atmosphere for productive work. Last but not least, I owe my loving thanks to my husband and lovely daughters who endured so much for me. And special gratitude goes to my Father and rest of my family members for their constant support and encouragement throughout my research work. And above all I thank Allah Finally, I thank all those who have helped me directly or indirectly in the successful completion of my thesis. Anyone missed in this acknowledgement are also thanked. 7 TABLE OF CONTENTS EXAMINATION COMMITTEE APPROVALS FORM ............................................................................. 2 ABSTRACT ......................................................................................................................................... 4 ACKNOWLEDGEMENTS .................................................................................................................... 6 TABLE OF CONTENTS........................................................................................................................ 7 LIST OF ABBREVIATIONS .................................................................................................................. 8 LIST OF ILLUSTRATIONS.................................................................................................................. 10 LIST OF TABLES ............................................................................................................................... 11 1. INTRODUCTION .......................................................................................................................... 12 1.1 Leukemia: Blockage in Hematopoietic differentiation ........................................................ 12 1.2 Differentiation therapy ........................................................................................................ 16 1.3 Phosphoproteomics and SILAC Labeling .............................................................................. 18 2. MATERIALS AND METHODS ..................................................................................................... 24 2.1 Cell Culturing and lysis ....................................................................................................... 24 2.2 Gel preparation and staining ............................................................................................... 26 2.3 Protein digestion and desalting......................................................................................... 27 2.4 Off-gel fractionation, Strong cation exchange chromatography and Phosphopeptide enrichment ................................................................................................................................. 28 Off-gel fractionation .................................................................................................................. 28 2.5 Mass spectrometric analysis and data processing ............................................................. 30 3. RESULTS...................................................................................................................................... 33 3.1 Quality of Lysate .................................................................................................................. 33 3.2 Protein Coverage of different lysate Portions: .................................................................... 35 3.3 Enrichment Efficiency .......................................................................................................... 38 3.4 Phosphorylated proteins found in Kg1a cells ...................................................................... 38 3.5 Phosphorylated Proteins found in HL60 cells ...................................................................... 43 4. DISCUSSION ................................................................................................................................ 45 BIBLIOGRAPHY ............................................................................................................................... 49 APPENDICES ................................................................................................................................... 52 8 LIST OF ABBREVIATIONS AML Acute Myeloid Leukemia AS2O3 Arsenic Trioxide BM Bone Marrow CAN Acetonitril CD34, CD38, CD44 Cluster of Differentiation Cys Cysteine Da Dalton DTT Dithiothreitol EGF Epidermal Growth Factor FBS Fetal Bovine Serum G-CSF Granulocyte Colony Stimulating Factor GO Gene ontology HAcO Acetic acid HPLC High Performance Liquid Chromatography HSC Hematopoietic stem cell IFNa Interferon alpha IMAC Immobilized Metal Affinity Chromatography IOA Iodoacetamide LC Liquid Chromatography LC-MS/MS Liquid Chromatography coupled with Mass Spectroscopy 9 LSC Leukemic Stem Cells mAb monoclonal Antibody MAPKKK Kinase in the Mitogen-Activated Protein Kinase pathway Met Methionine MS Mass spectroscopy pI Iso-electric point PTM Posttranslational modification pTyr phosphorylated Tyrosine SDS-PAGE Sodium Dodecyl Sulphate PolyAcrylamide Gel Electrophoresis Ser Serine SILAC Stable isotope labeling in Amino acid Culture TEAB Triethyl Ammonium Bicarbonate buffer TFA Trifluoroacetic acid Thr Threonine TiO2 Titanium dioxide Tyr Tyrosine 10 LIST OF ILLUSTRATIONS Figure 1.1: Conventional view of hematopoiesis .) ....................................................................... 14 Figure 1.2 Diagram of normal myeloid development and the relationship to leukemic cells and LSCs. Figure 1.3 Experimental outline for SILAC-based phophoproteomics. Figure 3.1 Gel Staining Proteins are loaded onto a SDS-page gels (4- 10 %) and subjected to SDS- PAGE electrophoresis. Figure 3.2: Flow diagram of the Phosphoproteomics platform applied ........................................ 37 Figure 3.3 Proportion of singly and multiply phosphorylated peptides in Kg1a cells. .................. 39 Figure 3.4 Distribution of identified phosphorylated serine, threonine, and tyrosine residues in resting Kg1a cells. .........................................................................................................................
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