Establishment of a two dimensional electrophoresis map of human mitochondrial proteins [Elektronische Ressource] / von Jing Xie

Jing Xie

Sujets

• Gesundheit

Informations

• Publié par: humboldt-universitat_zu_berlin
• Publié le: 01 janvier 2003
• Nombre de lectures: 50
• Poids de l'ouvrage: 7 Mo

Extrait

Aus der Klinik für Pädiatrie mit Schwerpunkt Neurologie
der Medizinischen Fakultät Charité der Humboldt-Universität zu Berlin



Dissertation

Establishment of a two-dimensional electrophoresis
map of human mitochondrial proteins


zur Erlangung des akademischen Grades
Doctor medicinae (Dr. med.)


vorgelegt der Medizinischen Fakultät Charité
der Humboldt-Universität Berlin



von
Jing XIE
geboren am 06.01.1970
aus Beijing (China)














Dekan der Medizinischen Fakultät Charité
Prof. Dr. J. W. Dudenhausen


Gutachter: 1. Prof. Dr. med. Markus Schülke-Gastenfeld
2. Prof. Dr. med. Thomas Meitinger
3. Prof. Dr. med. E. Wilichowski


DATUM DER PROMOTION: 15. 12. 2003

CONTENTS

ZUSAMMENFASSUNG ............................................................................................ VI
ABSTRACT .................................................................................................VIII
LIST OF ABBREVIATIONS ....................................................................................... X
LIST OF INTERNET SITES..................................................................................... XIII
1 INTRODUCTION .................................................................................................. 1
1.1 Introduction to mitochondria ........................................................................ 1
1.1.1 Mitochondrial morphology, biogenesis and composition........................... 1
1.1.2 Functions of the mitochondria................................................................... 1
1.1.2.1 Oxidative phosphorylation.................................................................................. 1
1.1.2.2 β-Oxidation......................................................................................................... 1
1.1.2.3 Citric acid cycle .................................................................................................. 2
1.1.2.4 Urea cycle ........................................................................................................... 2
1.1.2.5 Heme biosynthesis............................................................................................... 3
1.1.2.6 Apoptosis............................................................................................................. 4
1.1.3 Mitochondrial genetics .............................................................................. 4
1.1.4 Mitochondrial disorders............................................................................. 5
1.1.4.1 Definition of mitochondrial disorders ................................................................ 5
1.1.4.2 Classification of mitochondrial disorders .......................................................... 5
1.1.4.2.1 Mutations in the mtDNA ............................................................................... 5
1.1.4.2.2 Mutations in the nuclear DNA....................................................................... 6
1.1.4.3 Diagnosis of mitochondrial disorders 7
1.1.5 Characteristics of mitochondrial proteins and preproteins ........................ 7
1.2 Proteome analysis ......................................................................................... 8
1.2.1 Definition of proteome analysis................................................................. 8
1.2.2 Previous work on the proteome ................................................................ 9
1.3 The aim of my study ...................................................................................... 9
2 THEORY OF EMPLOYED METHODS............................................................... 11
2.1 Mitochondrial isolation................................................................................ 11
2.2 Determination of the protein concentration .............................................. 12
2.3 Two-dimensional electrophoresis techniques .......................................... 12
2.3.1 First dimension: isoelectric focussing ..................................................... 13
2.3.2 Second dimension: sodium dodecylsulfate (SDS)
I
-polyacrylamide gel electrophoresis........................................................ 13
2.3.3 Staining................................................................................................... 14
2.3.4 Reproducibility ........................................................................................ 14
2.4 Protein identification methods ................................................................... 14
2.4.1 Matrix-assisted laser desorption/ionization time-of-flight
mass spectrometry ................................................................................. 15
2.4.2 Peptide sequencing by MALDI-quadrupole time-of-flight
tandem mass spectrometry..................................................................... 17
2.4.3 Database search based on peptide mass fingerprint spectra ................. 17
3 MATERIALS AND METHODS ........................................................................... 19
3.1 Preparation of lymphoblastoid cell pellets................................................ 19
3.1.1 Chemicals and reagents ......................................................................... 19
3.1.2 Solutions................................................................................................. 19
3.1.3 Special equipment .................................................................................. 19
3.1.4 Procedure ............................................................................................... 19
3.1.4.1 Preparation of transformation medium ............................................................ 19
3.1.4.2 Preparation of mononuclear leukocytes from whole blood.............................. 20
3.1.4.3 Establishment of the permanent cell culture..................................................... 21
3.1.4.4 Preparation of the lymphoblastoid cell pellet................................................... 21
3.2 Preparation of mitochondria ....................................................................... 21
3.2.1 Chemicals and reagents ......................................................................... 21
3.2.2 Solutions................................................................................................. 21
3.2.3 Special equipment .................................................................................. 21
3.2.4 Procedure ............................................................................................... 22
3.2.4.1 Preparation of the post-nuclear supernatant.................................................... 22
3.2.4.2 Preparation of a hybrid Percoll/Metrizamide discontinuous gradient............. 22
3.2.4.3 Preparation of the mitochondrial pellet ........................................................... 23
3.3 Sample preparation of mitochondrial proteins ......................................... 23
3.3.1 Chemicals and reagents ......................................................................... 23
3.3.2 Solutions................................................................................................. 23
3.3.3 Special equipment .................................................................................. 24
3.3.4 Procedure ............................................................................................... 24
3.4 Bicinchoninic acid (BCA) protein assay .................................................... 24
3.4.1 Chemicals and reagents ......................................................................... 24
II
3.4.2 Special equipment .................................................................................. 24
3.4.3 Procedure ............................................................................................... 25
3.4.3.1 Preparation of diluted BSA serial standards .................................................... 25
3.4.3.2 Protein quantification assay ............................................................................. 25
3.5 Two-dimensional protein electrophoresis ................................................. 25
3.5.1 Chemicals and reagents ......................................................................... 25
3.5.2 Solutions................................................................................................. 26
3.5.3 Special equipment .................................................................................. 27
3.5.4 Procedure ............................................................................................... 27
3.5.4.1 First dimension-isoelectric focussing (IEF) ..................................................... 27
3.5.4.2 Sodium dodecyl-sulfate polyacrylamide gel electrophoresis............................ 28
3.5.4.3 Measurement of the pH-gradient of the IEF-gel............................................... 28
3.6 Gel staining and drying ............................................................................... 28
3.6.1 Chemicals and reagents ...............................................................