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Molecularly Imprinted Polymers for the Analysis of Protein Phosphorylation and the Role of Htra2/Omi Protein in Parkinson's Disease

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Molecularly Imprinted Polymers for the Analysis of Protein Phosphorylation and the Role of Htra2/Omi Protein in Parkinson's Disease Molecularly Imprinted Polymers for the Analysis of Protein Phosphorylation and the Role of HtrA2/Omi Protein in Parkinson's Disease by Jing Chen Dissertation Submitted to the Faculty of Chemistry and Biochemistry In Candidacy for the Degree of Doctor Rerum Naturalium (Dr. rer. nat) Accomplished at Medizinisches Proteom-Center Ruhr-Universität Bochum, Germany 03. 2015, Bochum Statement in Lieu of Oath I hereby declare that I have accomplished the thesis independently and did not submit to any other faculty or refer to more than the publications listed in the references. The digital figures contain only original data and no modification was added. There are altogether 5 identical copies of my dissertation. __________________________ Jing Chen I Referee: Prof. Dr. Katrin Marcus Co-referee: Dr. Dirk Wolters II Acknowledgement I would like to express my deep and sincere gratitude to Prof. Dr. Katrin Marcus, director of the Medizinische Proteom-Center, for her friendly invitation to the working group, for the great opportunity working in the interesting research field, for her dedication in supervising of my project execution and her unconditional help at the end of my Ph.D. I am very grateful to Dr. Dirk Wolters for his kind acceptance of attending and co- judging my dissertation. I owe my sincere gratitude to Dr. Stefan Helling, for his outstanding mentoring to this work. His valuable advice is deciding. Hadn’t for his endeavor in discussing and clearing my confusion at all times, I wouldn’t have managed to accomplish the work. I know Prof. Dr. Börje Sellergren, my collaboration partner at biomedical science in Malmö University, Sweden the longest. It was he who recommended me to work in MPC as a Ph.D candidate. I want to express my sincere gratitute to him for his constructive suggestions thoughout my Ph.D study. Dr. Sudhirkumar Shinde from Malmö University was very kind and helpful at work too. I want to thank him for the intensive discussion and his polymer material synthesis and characterization. Prabal Subedi contributed a lot in my research results discussion and dissertation correction. Kathy Pfeiffer performed many Western blotting for me. I am deeply grateful that she helped me so much during the stressful thesis-writing phase. I also want to thank Thilo Lerari, Sara Galozzi, Katalin Barkovits, Sarah Plum, Markus- Hermann Koch, Maike Ahrens, Caroline May, Jale Stoutjesdijk, Kathrin Barlog and all other MPC colleagues for reliable and whole-hearted assistance in my research, all Marie Curie PEPMIP members, for the discussions at project meetings and conferences. I thank Prof. Karl Mechtler from the Institute of Molecular Pathology (Vienna, Austria) and Mr. Ingo Feldmann from ISAS (Dortmund, Germany) for providing self- synthesized peptides, Rejko Krüger from Hertie-Institute for Clinical Brain Research at University Tuebingen for providing mouse brain and cell line sample material and David Just for his assistance raising cells in the cell culture. III In the end, I’d like to thank my beloved parents far away in China with all my heart. After years of striving in Germany I finally come this far. Without their support I couldn’t have made it. My boyfriend Judong Yang has been with me all these years by my side and supporting me so much from all aspects of life. Without him I am nowhere. I am truely grateful to have him experiencing those ups and downs in life together with me. IV Content Table 1. Introduction ....................................................................................................................... 1 1.1 Role of HtrA2/Omi in neurodegeneration and Parkinson's disease (PD) .......................... 1 1.1.1 HtrA2/Omi protein ......................................................................................................... 1 1.1.2 HtrA2/Omi and its mutations found in PD patients ........................................................ 3 1.1.3 Mouse model studies in relation to PD .......................................................................... 4 1.1.4 Cell stress model in relation to PD ................................................................................ 6 1.2 Protein phosphorylation and techniques for its analysis ................................................... 7 1.3 MIPs for phosphoanalysis ...............................................................................................10 1.4 Proteomics and applied MS techniques ..........................................................................13 1.5 Aims ...............................................................................................................................19 2. Materials and Methods .....................................................................................................20 2.1 Materials .........................................................................................................................20 2.1.1 Instruments, expendable items and chemicals ............................................................20 2.1.2 Buffers and reagents ...................................................................................................24 2.1.3 Antibodies ....................................................................................................................25 2.1.4 Abbriviation list ............................................................................................................25 2.2 Methods .........................................................................................................................26 2.2.1 Samples (cells, mouse brains, CSF) ............................................................................26 2.2.2 Cell culture and cell stress experiment ........................................................................28 2.2.3 Sample preparation .....................................................................................................28 2.2.4 Protein and peptide concentration determination .........................................................30 2.2.5 Western blots ..............................................................................................................31 2.2.6 Phosphopeptide enrichment ........................................................................................32 2.2.7 Instrumentation based analytical methods ...................................................................37 2.2.8 Label-free quantification with spectra counting ............................................................40 2.2.9 Data processing, databank search and pathway analysis ............................................41 2.2.10 Phosphopeptide motif-x analysis ...............................................................................42 3. Results .............................................................................................................................44 V 3.1 Efficiency comparison of pY-MIP, TiO2 and anti-pY antibodies for tyrosine phosphorylated peptides enrichment .............................................................................................................45 3.1.1 Method development and optimization ........................................................................45 3.1.2 Comparison results of PETRA and EDMA pY-MIPs ....................................................49 3.1.3 Comparison results of pY-MIP, TiO2 and anti-pY antibodies (standard peptides) .........50 1. Results of TiO2 SPE phosphopeptide enrichment .........................................................50 2. Results of phosphopeptide enrichment via three anti-pY antibodies .............................51 3. Deeper understanding of the conventional anti-pY antibodies ......................................53 4. Results of IP with combination of anti-pY antibodies .....................................................55 3.1.4 Comparison results of pY-MIP, TiO2 and anti-pY antibodies (spiking experiment) .......55 1. Results of pY-MIP performance ....................................................................................56 2. Results of TiO2 SPE performance .................................................................................56 3. Results of IP performance using 3 anti-pY antibodies ...................................................57 3.2 Results of pS-MIP phosphopeptide enrichment from biological samples ........................59 3.2.1 Template rebinding test ...............................................................................................59 3.2.2 Results of pS-MIP SPE sample loading and elution condition test ...............................61 3.2.3 Further probing for phosphopeptide recognition using pS-MIP ....................................63 3.2.4 Results of pS-MIP SPE targeting the single peptide spiked in mouse brain matrix ......64 3.2.5 Results of pS-MIP SPE application in human cell samples and comparison with TiO2 SPE ......................................................................................................................................70 3.2.6 Results of pS-MIP SPE application in clinical relevant samples ...................................74 3.3 Analysis of HtrA2/Omi model studies with novel methods in relation to PD ....................76 3.3.1 Results from global proteomics analysis of transgenic mouse brain samples ..............76 1. LC-MS/MS identified proteins with different ways of regulation .....................................76 2. G399S mutant HtrA2/Omi over-expression effect leading to 18 differential
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