Mass spectrometric characterization of elastin peptides and the effect of solar radiation on elastin [Elektronische Ressource] / von Melkamu Getie Kebtie

Sujets

• Biologie

Informations

• Publié par: martin-luther-universitat_halle-wittenberg
• Publié le: 01 janvier 2005
• Nombre de lectures: 51
• Langue: Deutsch
• Poids de l'ouvrage: 3 Mo

Extrait

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 02.10.1972 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:3-000009457
[http://nbn-resolving.de/urn/resolver.pl?urn=nbn%3Ade%3Agbv%3A3-000009457]Dedication














This work is dedicated to
my brother the late Dr. Anteneh Getie
and
my sister the late Ayalnesh Getie
IITable of contents
TABLE OF CONTENTS

1. INTRODUCTION................................................................................................................1
2. BACKGROUND ..................................................................................................................4
2.1. Elastic fibers .................................................................................................................4
2.1.1. Composition, structure and distribution .................................................................4
2.1.2. Biosynthesis of elastic fibers..................................................................................5
2.1.2.1. Tropoelastin synthesis ....................................................................................5
2.1.2.2. Elastic fiber assembly.....................................................................................7
2.1.2.3. Coacervation and cross-linking of tropoelastin............................................10
2.2. Elastic fibers in the skin.............................................................................................12
2.2.1. Skin aging.............................................................................................................13
2.2.2. Pathology..............................................................................................................16
2.3. Biochemical methods for elastin analysis.................................................................18
2.4. LC-MS of peptides and proteins...............................................................................19
2.5. Peptide and protein sequencing ................................................................................22
2.5.1. Edman degradation...............................................................................................22
2.5.2. Tandem mass spectrometry ..................................................................................22
2.5.3. De novo sequencing..............................................................................................26
2.5.4. Database searching ...............................................................................................27
3. RESULTS AND DISCUSSION ........................................................................................29
3.1. Evaluation of the effect of radiation on two elastin cross-links, desmosine and
isodesmosine (Appendix 6.1: Getie et al. 2003)......................................................29
3.2. Characterization of human skin elastin peptides (Appendix 6.2 and 6.3: Getie et
al. 2005a, Schmelzer et al. 2005)..............................................................................33
3.3. 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)........................37
3.4. Complementary mass spectrometric techniques to achieve complete sequence
coverage of recombinant human tropoelastin (Appendix 6.4: Getie et al. 2005b)
....................................................................................................................................38
3.4.1. Characterization of the peptic digest ....................................................................41
IIITable of contents
3.4.2. Characterization of chymotrypsin digest..............................................................41
3.4.3. Overall sequence coverage...................................................................................42
3.5. Cleavage sites of the enzymes used (Appendix 6.2 - 6.4: Getie et al. 2005a,
Schmelzer et al. 2005, Getie et al. 2005 b) ..............................................................42
3.6. Effect of UVB on recombinant human tropoelastin and human skin elastin
peptides......................................................................................................................43
3.7. Conclusive remark .....................................................................................................47
4. SUMMARY ........................................................................................................................48
5. REFERENCES...................................................................................................................54
6. APPENDIX (LIST OF PUBLICATIONS) ......................................................................64
6.1 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) 81-87.
6.2 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) 649-657.
6.3 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) 120-126.
6.4 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) 2989-2993.


IVAbbreviations
ABBREVIATIONS

AGE Glycation end products
amu Atomic mass unit
APCI Atmospheric pressure chemical ionization
API Atmospheric pressure ionisation
CI Chemical ionization
CIDCollision-induceddissociation
DCDirectcurrent
DEDelayedextraction
DES Desmosine
DESU Degradation product of DES with m/z 481.1 481
DESUm/z 497.1 497
ECM Extracellular matrix
EIElectronionization
ESIElectrosprayionization
FAB Fast atom bombardment
FT-ICR Fourier transform-ion cyclotron resonance
HP Hematoporphyrin
HPLC High performance liquid chromatography
IDEIsodesmosine
IDEU Degradation product of IDE with m/z 481.1 481
IDEUm/z 497.1 497
IOXD Isooxodesmosine
LC Liquid chromatography
MAGP Microfibril-associated glycoprotein
MALDI Matrix-assisted laser desorption ionization
MP Multiple positions
mRNA Messenger RNA
MSMassspectrometry
NMSC Nonmelanoma skin cancer
OXD Oxodesmosine
PDPlasma desorption
VAbbreviations
PSD Post-source decay
PTMPost-translationalmodification
qTOFquadrupoleTime-of-flight
RER Rough endoplasmic reticulum
RER i
rf Radio frequency
RNA Ribonucleic acid
ROS Reactive oxygen species
RP Reverse phase
TFA Triflouroacetic acid
TOF Time-of-flight
TSPThermospray
UVUltraviolet
VIIntroduction
1. INTRODUCTION

Ultraviolet (UV) radiation can be both beneficial and harmful to normal human skin. The
beneficial effects comprise killing pathogens on the skin, inducing vitamin D synthesis and
treating certain skin diseases such as psoriasis vulgaris. The harmful effects of UV radiation
are immune suppression, photoaging and, above all, skin carcinogenesis. The incidence of
skin cancer has been increasing at an astonishing rate over the past several decades due to
changes in leisure-time activity accompanied by an increased exposure to UV radiation. It is
estimated that more than one million new cases of nonmelanoma skin cancer (NMSC) occur
each year in the USA alone (Gloster and Brodland, 1996). The skin responds to sun exposure
by tanning and thickening, both of which provide some protection from further damage by
UV radiation. The degree of pigmentation in the skin and the ability to tan are important risk
factors in skin cancer development, and the risk of NMSC is highest in people who sunburn
easily and rarely tan (Gloster and Brodland, 1996).

Since most of the UV sunlight reaching the earth’s surface (>95%) is in the deeply
penetrating UVA (320–400 nm) region, solar radiation-induced photo-oxidative stress
effectively reaches into the human dermis (Sander, et al., 2002). The dermal elastic fibers
increase and become amorphous, a characteristic feature of a disease known as solar
elastosis. The clinical symptoms include dryness (roughness), irregular pigmentation,
wrinkling, elastosis and dilation of pre-existing blood vessels creating