MASS SPECTROMETRIC CHARACTERIZATION OF ELASTIN PEPTIDES AND THE EFFECT OF SOLAR RADIATION ON ELASTIN. Dissertation

Transcription

1 MASS SPECTROMETRIC CHARACTERIZATION OF ELASTIN PEPTIDES AND THE EFFECT OF SOLAR RADIATION ON ELASTIN Dissertation zur Erlangung des akademischen Grades Doktor rerum naturalium (Dr. rer. nat.) vorgelegt der Mathematisch-Naturwissenschaftlich-Technischen Fakultät (mathematisch-naturwissenschaftlicher Bereich) der Martin-Luther-Universität Halle-Wittenberg von Melkamu Getie Kebtie geboren am in Gundewoin, Äthiopien Gutachter: 1. Prof. Dr. Dr. Reinhard Neubert 2. Prof. Dr. Michael Linscheid 3. PD Dr. Johannes Wohlrab Halle (Saale), den 08. Dezember 2005 urn:nbn:de:gbv: [

2 Dedication This work is dedicated to my brother the late Dr. Anteneh Getie and my sister the late Ayalnesh Getie II

3 Table of contents TABLE OF CONTENTS 1. INTRODUCTION BACKGROUND Elastic fibers Composition, structure and distribution Biosynthesis of elastic fibers Tropoelastin synthesis Elastic fiber assembly Coacervation and cross-linking of tropoelastin Elastic fibers in the skin Skin aging Pathology Biochemical methods for elastin analysis LC-MS of peptides and proteins Peptide and protein sequencing Edman degradation Tandem mass spectrometry De novo sequencing Database searching RESULTS AND DISCUSSION Evaluation of the effect of radiation on two elastin cross-links, desmosine and isodesmosine (Appendix 6.1: Getie et al. 2003) Characterization of human skin elastin peptides (Appendix 6.2 and 6.3: Getie et al. 2005a, Schmelzer et al. 2005) Identification of the potential sites of proline hydroxylation in human skin elastin (Appendix 6.2 and 6.3: Getie et al. 2005a, Schmelzer et al. 2005) Complementary mass spectrometric techniques to achieve complete sequence coverage of recombinant human tropoelastin (Appendix 6.4: Getie et al. 2005b) Characterization of the peptic digest...41 III

4 Table of contents Characterization of chymotrypsin digest Overall sequence coverage Cleavage sites of the enzymes used (Appendix : Getie et al. 2005a, Schmelzer et al. 2005, Getie et al b) Effect of UVB on recombinant human tropoelastin and human skin elastin peptides Conclusive remark SUMMARY REFERENCES APPENDIX (LIST OF PUBLICATIONS) Getie, M.; Raith, K.; Neubert, H.H.R., LC/ESI-MS analysis of two elastin cross-links, desmosine and isodesmosine, and their radiation-induced degradation products, Biochim Biophys Acta, 1624 (2003) Getie, M.; Schmelzer, C; Neubert, H.H.R., Characterization of peptides resulting from digestion of human skin elastin with elastase, Proteins: Structure, Function, and Bioinformatics, 61 (2005a) Schmelzer, C; Getie, M.; Neubert, H.H.R., Mass spectrometric characterization of human skin elastin peptides produced by proteolytic digestion with pepsin and thermitase, J Chromatogr A, 1083 (2005) Getie, M.; Schmelzer, C.; Weiss, A.; Neubert, H.H.R., Application of complementary mass spectrometric techniques to achieve a complete sequence coverage of recombinant human tropoelastin, Rapid Commun Mass Spectrom, 19 (2005b) IV

5 Abbreviations ABBREVIATIONS AGE Glycation end products amu Atomic mass unit APCI Atmospheric pressure chemical ionization API Atmospheric pressure ionisation CI Chemical ionization CID Collision-induced dissociation DC Direct current DE Delayed extraction DES Desmosine DESU 481 Degradation product of DES with m/z DESU 497 Degradation product of DES with m/z ECM Extracellular matrix EI Electron ionization ESI Electrospray ionization FAB Fast atom bombardment FT-ICR Fourier transform-ion cyclotron resonance HP Hematoporphyrin HPLC High performance liquid chromatography IDE Isodesmosine IDEU 481 Degradation product of IDE with m/z IDEU 497 Degradation product of IDE with m/z IOXD Isooxodesmosine LC Liquid chromatography MAGP Microfibril-associated glycoprotein MALDI Matrix-assisted laser desorption ionization MP Multiple positions mrna Messenger RNA MS Mass spectrometry NMSC Nonmelanoma skin cancer OXD Oxodesmosine PD Plasma desorption V

6 Abbreviations PSD PTM qtof RER RER rf RNA ROS RP TFA TOF TSP UV Post-source decay Post-translational modification quadrupole Time-of-flight Rough endoplasmic reticulum Rough endoplasmic reticulum Radio frequency Ribonucleic acid Reactive oxygen species Reverse phase Triflouroacetic acid Time-of-flight Thermospray Ultraviolet VI