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Proteomic Analysis of Ocular Surface Components by Use of HPLC Based Mass Spectrometric Strategies

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Proteomic Analysis of Ocular Surface Components by Use of HPLC Based Mass Spectrometric Strategies Proteomic Analysis of Ocular Surface Components by use of HPLC based Mass Spectrometric Strategies Dissertation zur Erlangung des Grades „Doktor der Naturwissenschaften“ am Fachbereich Biologie der Johannes Gutenberg-Universität in Mainz von Sebastian Funke geboren in Iserlohn Mainz, 15.09.2014 Dekan: 1. Berichterstatter: 2. Berichterstatter: Tag der mündlichen Prüfung: 20.04.2015 Annotation Parts of the thesis have been published in international journals and/or presented on international conferences Publications related to the thesis Funke, S., Azimi, D., Wolers, D, Grus, F. H, Pfeiffer, N. 2012. Longitudinal analysis of taurine induced effects on the tear proteome of contact lens wearers and dry eye patients using a RP-RP-Capillary HPLC-MALDI TOF/TOF MS approach. Journal of Proteomics 75: 3177- 3190 Bell, K., Funke, S., Pfeiffer, N., and Grus, F.H. 2012. Serum and antibodies of glaucoma patients lead to changes in the proteome, especially cell regulatory proteins, in retinal cells. PLoS One 7(10): e46910 Bell, K., Gramlich, O. W., Von Thun Und Hohenstein-Blaul, N., Beck, S., Funke, S., Wilding, C.; Pfeiffer, N., Grus, F. H. 2013. Does autoimmunity play a part in the pathogenesis of glaucoma? Progress in Retinal and Eye Research 36: 199-216 Von Thun Und Hohenstein-Blaul, N., Funke, S., Grus, F.H. 2013. Tears as a source of biomarkers for ocular and systemic diseases. Experimental Eye Research. 117: 126-137 Boehm, N., Funke, S., Wiegand, M., Wehrwein, N., Pfeiffer, N., and Grus, F.H. 2013. Alterations in the tear proteome of dry eye patients--a matter of the clinical phenotype. Investigative Ophthalmolology & Vision Science 54(3): 2385-2392 Perumal, N., Funke, S., Pfeiffer, N., Grus, F. H. 2014. Characterization of lacrimal proline- rich protein 4 (PRR4) in human tear proteome. Proteomics 00: 1-12 Bell, K., Wilding, C., Funke, S., Pfeiffer, N., Grus, F. H. 2014. Protective effect of 14-3-3 antibodies on stressed neuroretinal cells via the mitochondrial pathway. BMC Neuroscience, submitted Perumal, N., Funke, S., Wolters, D., Pfeiffer, N., Grus, F. H. 2014. Upregulation of lacrimal proline-rich protein 4 (PRR4) in human reflex tear proteome. Proteomics, submitted Funke, S., Beck, S., Lorenz, K., Kotterer, M., Wolters, D., Pfeiffer, N., Grus, F. H. 2014. Analysis of the effects of preservative-free tafluprost on the tear proteome. Opthalmology, submitted I Publications related to the thesis (in preparation) Funke, S., Bell, K., Wilding, C., Wolters, D., Pfeiffer, N., Grus, F. H. Taurine effects on the conjunctival cell proteome. Funke, S., Bechter, L., Perumal, N., Wolters, D., Pfeiffer, N., Grus, F. H. Proteomic analysis of glaucomatous human trabecular meshwork. Funke, S., Perumal, N., Gabelt-Scheurich, S., Gramlich, O., Beck, S., Boehm, N., Wolters, D., Pfeiffer, N., Grus, F. H. Proteomic mass spectrometric analysis of the human glaucomatous retina. Funke, S., Perumal, N., Scieranski, M., Beck, S., Pfeiffer, N., Grus, F. H. Proteomic investigation on the tear proteome of glaucoma patients with dry eye sensation. Funke, S., Perumal, N., Gabelt-Scheurich, S., Mwiiri, F. K., Wolters, D, Gerbig, C., Weiler, L., Pfeiffer, N., Grus, F. H. In depth mass spectrometric analyis of the porcine retina proteome. Perumal, N., Funke, S., Wolters, D., Pfeiffer, N., Grus, F. H. Identification of human tear fluid biomarkers in different dry eye disease subgroups using label-free quantitative proteomics. Poster presentations on international conferences Funke, S., Azimi, D., Pfeiffer, N., Grus F. H. 2011. Taurine induced effects on the tear proteome of dry eye patients and contact lens wearers using a RP-RP-2D-capillary-LC- MALDI-TOF/TOF MS approach. 6680-D1167. Fort Lauderdale, USA. ARVO Visionary Genomics World Conference 2011 Perumal, N., Funke, S., Boehm, N., Peiffer, N., Grus, F. H. 2011. Defining proline rich proteins (PRPs) distribution in tear proteome. 6681-D1168. Fort Lauderdale, USA. ARVO Visionary Genomics World Conference 2011 Wolters, D., Funke, S., Grus, F. H., Pfeiffer, N. 2012. Post transcriptional modifications in mass spectrometry based proteomics data. P8. Obergurgl, Austria. ARVO Optic Nerve Degeneration and Ageing Conference 2012. Wilding, C., Bell, K., Funke, S., Pfeiffer, N., Grus, F. H. 2012. Gamma synuclein antibody effects on mitochondrial apoptosis pathways. P2. Obergurgl, Austria. ARVO Optic Nerve Degeneration and Ageing Conference 2012. II Wilding, C., Bell, K., Funke, S., Beck, S., Peiffer, N., Grus, F. H. 2013. Protective effect of GFAP antibody on RGC5 cells via actin cytoskeleton pathways. 432-A0163. Seattle, USA. ARVO Life Changing Research World Conference 2013 Funke, S., Wolters, D., Weiler, L., Mwiiri, F. K., Bell, K., Wilding, C., Peiffer, N., Grus, F. H. 2013. In depth analysis of retinal proteins using a combinatory HPLC based mass spectrometric platform. 698-D0255. Seattle, USA. ARVO Life Changing Research World Conference 2013 Bell, K., Wilding, C., Funke, S., Peiffer, N., Grus, F. H. Changes in antibodies from glaucoma patients lead to changes in apoptosis pathways of neuroretinal cells. 1605-D0032. Seattle, USA. ARVO Life Changing Research World Conference 2013 Perumal, N., Funke, S., Pfeiffer, N., Grus, F. H. Characterization and post-translational modifications of proline-rich protein 4 (PRP4) in basal and reflex tear proteome of human. 2453-D0058. Seattle, USA. ARVO Life Changing Research World Conference 2013 Perumal, N., Funke, S., Pfeiffer, N., Grus, F. H. 2013. Significant up-regulation of proline-rich protein 4 (PRR4) in human reflex tear proteome. St Malo, France. EuPA Conference 2013 Funke, S., Lorenz, K., Kotterer, M., Beck, S., Wolters, D., Pfeiffer, N., Grus, F. H. 2014. Analysis of the effects of Taflotan® sine on tear protein profiles. 1474-C0113. Orlando, USA. ARVO Leading Eye and Vision Research World Conference 2014 Perumal, N., Funke, S., Wolters, D., Pfeiffer, N., Grus, F. H. 2014. In depth protein profiling and identification of tear fluid biomarkers in different subgroups of dry eye disaese: Proline- rich protein 4 (PRR4) as apotential biomarker for aqueous-deficient dry eye syndrome. 2002- B0301. Orlando, USA. ARVO Leading Eye and Vision Research World Conference 2014 Wilding, C., Bell, K., Funke, S., Pfeiffer, N., Grus, F. H. 2014. Participation of ERP57 in the pro-survival potential of GFAP antibodies on RGC5. 2438-B0137. Orlando, USA. ARVO Leading Eye and Vision Research World Conference 2014 Perumal, N. , Funke, S., Pfeiffer, N., Grus, F. H. 2014. Identification of human tear fluid biomarkers in different dry eye disease subgroups using label-free quantitative proteomics. Madrid, Spain. 13th Human Proteome Organization World Congress, accepted III Oral papers on international conferences Funke, S., Beck, S., Boehm, N., Pfeiffer, N., Grus, F. H. 2012. Retinal biomarkers in glaucoma. O063. Berlin, Germany. ISER XX Biennial Meeting of the International Society for Eye Research Conference 2012 Funke, S., Wolters, D., Bell, K., Wilding, C., Pfeiffer, N., Grus, F. H. 2012. Protein modifications and ageing. Obergurgl, Austria. ARVO Optic Nerve Degeneration and Ageing Conference 2012. Funke, S., Bell, K., Wilding, C., Perumal, N., Pfeiffer, N, Grus, F. H. 2013. Mass spectrometric based strategies to recover and characterize proteins from retinal sample species. Obergurgl, Austria. Optic Nerve Conference 2013 Wilding, C., Bell, K., Beck, S., Funke, S., Pfeiffer, N., Grus, F. H. 2013. GFAP antibodies interact with ERP57 on the cell membrane of RGC5 cells and have prosurvival potential against oxidative stress. Obergurgl, Austria. Optic Nerve Conference 2013 Research prices Sicca-Förderpreis des Ressorts Trockenes Auge und Oberflächenerkrankungen im Berufsverband der Augenärzte Deutschlands 2013. Taurine induced effects on tear film and ocular surface revealed by mass spectrometric based proteomics. Berlin, Germany. 111th DOG Excellent Vision-Vision of Excellence Conference 2013 IV Aknowledgements First, I would like to thank my supervisor for the opportunity to realize the thesis in the Experimental Ophthalmology lab of the University Medical Center Mainz and for his help, support and scientific experience he shared with me. I also thank my co-supervisor for his support. Furthermore, I would like to thank my collegues for their contribution to the realization of this work. Special thanks go to my wife and families for their help and support over the past years. I Table of Content Table of Content Table of Content I 1 Purpose 1 2 Introduction 2 2.1 Mass spectrometry (MS) based proteomics ............................................................... 2 2.2 Bottom Up (BU) vs. Top Down (TD) ........................................................................... 4 2.3 MALDI-TOF-(TOF) MS (/MS) ..................................................................................... 6 2.4 LTQ-Orbitrap XL MS .................................................................................................. 8 2.5 HPLC in mass spectrometry ......................................................................................10 2.6 Ocular surface proteomics ........................................................................................12 2.7 Dry Eye: Causes and consequences ........................................................................15 2.8 Therapeutic management with special focus on Taflotan® sine and taurine ..............18 3 Material & Methods 23 3.1 Training samples .......................................................................................................23
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