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: 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

Zusammenfassung

Mitochondriopathien sind Multisystemerkrankungen die durch verschiedene Defekte in den Energie (ATP) produzierenden Stoffwechselwegen der Mitochondrien verursacht sind. Will man Mitochondriopathien auf molekularer Ebene diagnostizieren, stößt man auf folgende Schwierigkeiten: (A) Ungefähr 1000 Gene sind an der Biogenese des Mitochondriums beteiligt. Die Dysfunktion jedes einzelnen dieser Gene kann potentiell zur Mitochondriopathie führen. (B) Mitochondriale Proteine werden durch zwei Genome, durch die mitochondriale und durch die nukleäre DNA kodiert. (C) Die klinischen Symptome der Patienten weisen selten auf die molekulare Diagnose, da der Phänotyp oft nur auf einem sekundären Energiemangel beruht. In der Regel besteht keine sichere Genotyp-Phänotyp-Relation.

Mit den gegenwärtig zur Verfügung stehenden Methoden lassen sich bei nur 20% der Patienten Mutationen finden. Wir wollten daher eine neue Screening-Methode entwickeln, mit deren Hilfe wir hoffen, die Aufspürungsrate für mitochondriale Mutationen zu erhöhen. Die Gesamtheit der Proteine einer Organelle oder einer ganzen Zelle (ihr “Proteom”) stellt das Verbindungsglied zwischen Geno- und Phänotyp dar. Aus diesem Grunde wollten wir das mitochondriale Proteom von gesunden Kontrollpersonen und von Patienten mit Mitochondriopathien untersuchen. Protein-Muster, die zwischen diesen beiden Gruppen abweichen, könnten die Aufmerksamkeit auf Gene und Proteine richten, die an der Entstehung des Krankheits-Phänotyps beteiligt sind. Um solch eine vergleichende Studie durchzuführen, muß zunächst eine Referenzkarte des normalen mitochondrialen Proteoms erstellt werden. In meinem Dissertationsprojekt habe ich diese grundlegende Arbeit durchgeführt und zahlreiche Proteine auf der Proteomkarte menschlicher Mitochondrien identifiziert, die aus Epstein-Barr-Virus-transformierten lymphoblastoiden Zellen gewonnen worden waren. Ich wählte diese Zellsorte als Untersuchungsmaterial, da sie nicht nur einfach von Patienten gewonnen werden, sondern auch potentiell permanent wachsen kann. Dies erlaubt die Züchtung einer hohen Zellzahl ohne übermäßigen Aufwand. Ich optimierte ein Protokoll zur Zentrifugation in einem hybriden Gradienten, mit dem genug gereinigte Mitochondrien aus 10⁸ Zellen gewonnen werden konnten. Für die Referenzkarte benutzte ich die lymphoblastoide Zellline einer gesunden Kontrollperson.

Die Methode der Wahl zur Proteinidentifikation in Proteom-Projekten ist die zweidimensionale Proteinelektrophorese gekoppelt mit der MALDI-TOF-Massenspektrometrie. Ich entdeckte mehr als 400 Punkte in meinem silbergefärbten zweidimensionalen Gel und analysierte die 141 stärksten Punkte nach in-gel Trypsin-Verdau [page vii↓] und anschließender MALDI-TOF-Massenspektrometrie in einem Verfahren, das als “Peptide Mass Fingerprinting” (Peptidmassen-Fingerabdruck) bezeichnet wird. Mit Hilfe entsprechender Datenbanken konnte ich schließlich 115 verschiedene Proteinpunkte (entsprechen 95 verschiedenen Proteinen) identifizieren. 90 dieser Punkte (entsprechend 74 verschiedenen Proteinen) waren sicher mitochondrialer Herkunft und sind Komponenten aller wesentlichen im Mitochondrium lokalisierten Stoffwechselwege. 16 der 74 identifizierten mitochondrialen Proteine gehören zur Atmungskette. Obwohl 18 mitochondriale Proteine in der Datenbank SWISS-PROT als “Membran-assoziiert” annotiert sind, identifizierte ich nur vier Proteine mit sicheren Transmembrandomänen. Ich entdeckte keine der 13 durch die mitochondriale DNA kodierten Proteine, die alle stark hydrophobe Membranproteine sind. Andere Forscher sind bei dem Versuch diese Proteine zu identifizieren, auf die gleichen Schwierigkeiten gestoßen.

Mit meiner Dissertationsarbeit habe ich unsere eigene Datenbank und Referenzkarte des mitochondrialen Proteoms lyphoblastoider Zellen erstellt. Diese Daten ermöglichen nun die Analyse des mitochondrialen Proteoms von Patienten. Meine weiteren Untersuchungen auf diesem Gebiet werden sich nun auf die genetische Variabilität des Proteoms gesunder Kontrollpersonen und auf das Proteom der Patienten mit Mitochondriopathien beziehen.

Eigene Schlagworte: Mitochondrien, Proteom, Dichtegradientenzentrifugation, zweidimensionale Proteinelektrophorese, Proteinidentifikation, MALDI-TOF Massenspektrometrie, Peptidmassen-Fingerabdruck

Abstract

Mitochondrial disorders are multisystem diseases that can be caused by any defect in the energy (ATP) generating pathways in the mitochondria. The difficulty in diagnosing mitochondrial diseases on the molecular level arises from several obstacles: (A) About 1000 genes are involved in the biogenesis of mitochondria. The dysfunction of each of them may potentially cause mitochondriopathy. (B) The mitochondrial proteins are encoded by two genomes: the mitochondrial DNA and the nuclear DNA. (C) The clinical symptoms of the patients rarely suggest a molecular diagnosis since in most cases, the phenotype is a secondary phenomenon to energy depletion. Generally there is no genotype-phenotype relation.

Based on current diagnostic methods in only 20% of the patients a mutation can be found. We therefore wanted to develop a new screening method by which we hope to increase the identification rate. Since the numerous proteins of an organelle or of a whole cell (its “proteome”) connect the genotype with the phenotype, we set out to study the proteome of the mitochondrion in healthy individuals and in patients with mitochondrial diseases. Deviating protein patterns between the two individuals could direct the attention to disease-specific proteins and genes, which might be involved in the expression of a disease-phenotype. In order to perform such a comparison I first had to establish a normal reference map. In my dissertation project I performed this basic task and identified numerous mitochondrial proteins on the proteome-map of human mitochondria, which had been extracted from lymphoblastoid cells. I selected Epstein-Barr-Virus-transformed lymphoblastoid cells as samples not only because they are easily obtained from patients, but also due to their potential permanent growth. This approach allows the cultivation of high cell numbers without excessive expenditure of work and cost. I optimized a protocol for hybrid gradient centrifugation, by which enough mitochondria can be purified from 10⁸ cells. I used a cultured lymphoblastoid cell line from a normal control patient and isolated mitochondria from it by using hybrid gradient centrifugation. In proteomics the combination of the high-resolution two-dimensional electrophoresis and matrix assisted laser desorption/ionization–time–of–flight–mass spectrometry (MALDI-TOF-MS) is currently the method of choice for protein identification. I detected more than 400 spots in a silver-stained two-dimensional gel. I analyzed the 141 strongest spots of it by trypsin in gel digestion and subsequent MALDI-TOF mass spectrometry in a process termed “peptide mass fingerprinting”. After database search, I finally identified 115 protein spots (corresponding to [page ix↓]95 different proteins), 90 of which (corresponding to 74 different proteins) are of confirmed mitochondrial origin. These identified proteins are components of the main biological pathways located in the mitochondrion. 16 of the 74 identified mitochondrial proteins belong to the respiratory chain. Despite the fact that 18 mitochondrial proteins are annotated in the SWISS-PROT-database as “membrane associated proteins”, only four of them have clear transmembrane domains. None of the proteins encoded by the mitochondrial DNA could be detected. All of them are hydrophobic membrane proteins. A similar difficulty in resolving these proteins was encountered by other research groups.

With my dissertation I established our own database and reference map of the mitochondrial proteome of lymphoblastoid cells. These data will facilitate the analysis of the mitochondrial proteome in patients. My future research based on this dissertation will mainly focus on the genetic variation of the proteome of healthy individuals and on patients with mitochondrial diseases.

Keywords: mitochondria, proteome, density gradient centrifugation, two-dimensional protein electrophoresis, protein identification, MALDI-TOF mass spectrometry, peptide mass fingerprinting

Table of contents

• List of Abbreviations
• 1.  Introduction
  • 1.1. Introduction to mitochondria
    • 1.1.1. Mitochondrial morphology, biogenesis and composition
    • 1.1.2. Functions of the mitochondria
      • 1.1.2.1. Oxidative phosphorylation
      • 1.1.2.2. β-Oxidation
      • 1.1.2.3. Citric acid cycle
      • 1.1.2.4. Urea cycle
      • 1.1.2.5.  Heme biosynthesis
      • 1.1.2.6.  Apoptosis
    • 1.1.3. Mitochondrial genetics
    • 1.1.4. Mitochondrial disorders
      • 1.1.4.1. Definition of mitochondrial disorders
      • 1.1.4.2. Classification of mitochondrial disorders
        • Mutations in the mtDNA
        • Mutations in the nuclear DNA
      • 1.1.4.3. Diagnosis of mitochondrial disorders
    • 1.1.5. Characteristics of mitochondrial proteins and preproteins
  • 1.2. Proteome analysis
    • 1.2.1. Definition of proteome analysis
    • 1.2.2. Previous work on the proteome
  • 1.3. The aim of my study
• 2.  Theory of employed methods
  • 2.1. Mitochondrial isolation
  • 2.2. Determination of the protein concentration
  • 2.3. Two-dimensional electrophoresis techniques
    • 2.3.1. First dimension: isoelectric focussing
    • 2.3.2. Second dimension: sodium dodecylsulfate (SDS)-polyacrylamide gel electrophoresis
    • 2.3.3. Staining
    • 2.3.4. Reproducibility
  • 2.4. Protein identification methods
    • 2.4.1. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry
    • 2.4.2.  Peptide sequencing by MALDI-quadrupole time-of-flight tandem mass spectrometry
    • 2.4.3. Database search based on peptide mass fingerprint spectra
• 3.  Materials and methods
  • 3.1. Preparation of lymphoblastoid cell pellets
    • 3.1.1. Chemicals and reagents
    • 3.1.2. Solutions
    • 3.1.3. Special equipment
    • 3.1.4. Procedure
      • 3.1.4.1. Preparation of transformation medium
      • 3.1.4.2. Preparation of mononuclear leukocytes from whole blood
      • 3.1.4.3. Establishment of the permanent cell culture
      • 3.1.4.4. Preparation of the lymphoblastoid cell pellet
  • 3.2. Preparation of mitochondria
    • 3.2.1. Chemicals and reagents
    • 3.2.2. Solutions
    • 3.2.3. Special equipment
    • 3.2.4. Procedure
      • 3.2.4.1. Preparation of the post-nuclear supernatant
      • 3.2.4.2. Preparation of a hybrid Percoll/Metrizamide discontinuous gradient
      • 3.2.4.3. Preparation of the mitochondrial pellet
  • 3.3.  Sample preparation of mitochondrial proteins
    • 3.3.1. Chemicals and reagents
    • 3.3.2. Solutions
    • 3.3.3. Special equipment
    • 3.3.4. Procedure
  • 3.4. Bicinchoninic acid (BCA) protein assay
    • 3.4.1. Chemicals and reagents
    • 3.4.2. Special equipment
    • 3.4.3.  Procedure
      • 3.4.3.1. Preparation of diluted BSA serial standards
      • 3.4.3.2. Protein quantification assay
  • 3.5. Two-dimensional protein electrophoresis
    • 3.5.1. Chemicals and reagents
    • 3.5.2. Solutions
    • 3.5.3. Special equipment
    • 3.5.4. Procedure
      • 3.5.4.1. First dimension-isoelectric focussing (IEF)
      • 3.5.4.2. Sodium dodecyl-sulfate polyacrylamide gel electrophoresis
      • 3.5.4.3. Measurement of the pH-gradient of the IEF-gel
  • 3.6. Gel staining and drying
    • 3.6.1. Chemicals and reagents
    • 3.6.2.  Solutions
    • 3.6.3. Special equipment and material
    • 3.6.4. Procedure
      • 3.6.4.1. Silver staining
      • 3.6.4.2.  Colloidal Coomassie staining
      • 3.6.4.3. Gel drying and preserving
  • 3.7. Sample preparation for MALDI-TOF protein mass fingerprinting
    • 3.7.1. Chemicals and reagents
    • 3.7.2. Solutions
    • 3.7.3. Special equipment
    • 3.7.4. Procedure
      • 3.7.4.1. In-gel digestion
      • 3.7.4.2. Sample preparation for MALDI analysis
  • 3.8. Peptide mass fingerprinting by MALDI-TOF mass spectrometry
    • 3.8.1. Special equipment
    • 3.8.2. Procedure
  • 3.9. Computer aided analysis of protein mass fingerprints
  • 3.10. Peptide sequencing by MALDI-QTOF mass spectrometry
    • 3.10.1. Chemicals and reagents
    • 3.10.2. Solutions
    • 3.10.3. Special equipment
    • 3.10.4. Procedure
      • 3.10.4.1. Sample purification by nano-scale reversed-phase chromatography
      • 3.10.4.2. Protein ladder sequencing of peptide fragments
• 4.  Results
  • 4.1. Mitochondrial isolation
  • 4.2. Preparation of protein samples
  • 4.3. 2D-electrophoresis of mitochondrial proteins
    • 4.3.1. The pH-gradient of the IEF-gel
    • 4.3.2. Two-dimensional electrophoresis
    • 4.3.3. Gel staining
    • 4.3.4.  Influence of repeated freezing-thawing cycle on sample quanlity
  • 4.4. Protein identification
    • 4.4.1. MALDI-TOF and MALDI-QTOF tandem mass spectrometry
    • 4.4.2. Database search for protein identification
  • 4.5. Mitochondrial proteome reference map
    • 4.5.1. Mitochondrial proteome reference map
    • 4.5.2. Locations of the identified proteins
    • 4.5.3. Functions of the identified mitochondrial proteins
    • 4.5.4. Identified membrane proteins
    • 4.5.5. Multiple spot proteins
    • 4.5.6. Comparison of theoretical and the experimental pI and MW
• 5.  Discussion
  • 5.1. Choice of material
  • 5.2. Mitochondrial isolation
  • 5.3. Two-dimensional electrophoresis
    • 5.3.1. The choice of carrier ampholytes for isoelectric focussing.
    • 5.3.2. Reproducibility
    • 5.3.3. The number of the visualized proteins on the gel
    • 5.3.4. Staining of the gel
  • 5.4. Protein identification
  • 5.5. The mitochondrial proteome reference map
    • 5.5.1. The identified membrane proteins
    • 5.5.2. Multiple spots proteins
    • 5.5.3. Comparison of my results with other mitochondrial proteomic projects
    • 5.5.4. Comparison of the theoretical and the experimental pI and MW
      • 5.5.4.1. Comparison of the experimental pI and the theoretical pI
      • 5.5.4.2. Comparison of the experimental MW and the theoretical MW
• 6. Concluding remarks
• Supplementary material
• References
• Acknowledgments
• Curriculum Vitae
• Erklärung

Tables

• Tab. 4-1 : Sorting of the identified proteins according to their subcellular locations and functions. Proteins with transmembrane domains predicted by the SOSUI-algorithm are listed and highlighted separately.
• Tab. 4-2: Classification of the identified proteins according to their location. The mitochondrial group (M) includes those proteins that are definitively mitochondrial but for which no further information exists on their exact location.
• Tab. 5-1: Comparison of carrier ampholytes and immobilized pH-gradients for first dimension isoelectric focussing.
• Tab. 7-1: The identified proteins of my study. Proteins from the mitochondrial fraction of human lymphoblastiod cells were extracted and separated by 2D-electrophoresis. The identification of proteins was carried out with MALDITOF and MALDI-QTOF mass spectrometry. The database search was performed with several search engines such as Mascot, ProFound, and MS-Fit. Information about a specific protein contains the spot number, the full name, the accession ID and SWISS-PROT number, the E.C. and gi numbers, the theoreticalMW and pI values from SWISS-PROT database [MW(S) and pI(S)] and the measuredMW and pI values [MW(m) and pI(m)].The data from the MS analysis i.e. the numbers of matching peptides and the probability of assignment of a random identity are listed here as well. M: mitochondrial; MM: mitochondrial matrix; MIM: mitochondrial inner membrane; MOM: mitochondrial outer membrane; MIMS: mitochondrial inter membrane space.

Images

• Fig. 1-1: The citric acid cycle is the final common pathway for the oxidation of fuel molecules. Most fuel molecules enter the cycle as acetyl-CoA. The cycle starts with the fusion of oxaloacetate and acetyl-CoA to citrate. Citrate then undergoes a series of isomerisation-, oxidation-, and decarboxylation-steps that finally regenerate oxaloacetate. The free energy of these intermittent steps is used for the reduction of three molecules NAD+ and one molecule FAD+ . The NADH- and FADH2 -molecules thus generated, are subsequently delivered to the respiratory chain of the mitochondrion to generate ATP.
• Fig. 1-2: The urea cycle is part of the degradation pathway of amino acids. It converts the NH4 + generated by amino acid degradation into urea. The first reaction of the urea cycle ― the condensation of ornithine and carbamaylphosphate - takes place in the mitochondrial matrix. Citrulline is then exported into the cytosol.
• Fig. 1-3: The heme biosynthesis occurs partly in the mitochondrion and partly in the cytoplasm. The first step (the condensation of succinyl-CoA and glycine to δ- aminolevulic acid) and the final two steps (production of heme) take place in the mitochondrion. Most of the intermediate steps take place in the cytoplasm.
• Fig. 1-4: Preprotein import pathways into the mitochondrion . Before being transported into the mitochondrion, proteins are synthesized as preproteins in the cytosol. They are then imported through the translocases of the outer membrane (TOM). Preproteins with a typical amino-terminal targeting sequence engage with the inner-membrane (TIM) complex 17+23 (pathway A) to be imported into the matrix. In the matrix the targeting sequences are removed by the matrix-processing protease to form the mature proteins. Preproteins, which lack a targeting sequence, engage with the TIM22 complex (pathway B) to be inserted into the inner membrane.
• Fig. 2-1: Principle of 2D-electrophoresis . In 2D-electrophoresis a complex protein mixture can be separated by two biochemical principles. In the first dimension isoelectric focussing (IEF) the proteins are separated according to their isoelectric points (pI ), e.g. proteins run in an electric field as long as the surrounding pH differs from their pI . If they reach their pI , their net charge is zero and they stop running in the electric field. In the second dimension, proteins are separated according to their molecular weights (MW ) in a SDS-polyacrylamide gel.
• Fig. 2-2 : Principle of peptide mass fingerprinting by trypsin digestion . Trypsin cleaves at the carboxylic side of arginine and lysine residues. The sizes of the peptide fragments obtained after trypsin digestion, represent the peptide mass fingerprint and are characteristics of each protein.
• Fig. 2-3 : Principle of MALDI-TOF (matrix-assisted laser desorption/ionization time-of-flight) mass spectrometry . The peptides in the sample are ionized and energized by a laser-beam and are accelerated in an electric field. The time of flight (=TOF) of each peptide fragment towards a target is measured. Since the TOF is proportional to the mass/charge ratio of each peptide, the mass of the peptide can thus be calculated. This way the mass spectra of the peptide fragments of a whole protein can be obtained.
• Fig. 2-4: Principle of MALDI-QTOF (quadrupole time-of-flight) tandem mass spectrometry . The sample is ionized and energized by a laser-beam and flies into a quadrupole ion guiding cell (Q0 ), where the ions are focussed and cooled. Then a peptide of interest (the parent ion) is selected at the quadrupole Q1 -cell and guided into the quadrupole collision cell (Q2 ). There the parent ion collides with argon atoms and splits into daughter-ions. The masses of these daughter ions are then measured via TOF mass spectrometry.
• Fig. 3-1: Establishment of lymphoblastoid cell lines . The left column depicts the preparation of the transformation medium. The right column depicts the preparation of the mononuclear cells. The final bottle contains both the transformation medium and the mononuclear cells, which are set up for cultivation of a lymphoblastoid cell line.
• Fig. 3-2 : Mitochondrial isolation by Percoll/metrizamide hybrid gradient centrifugation. Electron microscopy reveals that the first fraction contains mostly membrane debris from both cellular and subcellular organelles. The material of the second fraction contains a few lysosomes and other structures that are difficult to identify morphologically. Only the third fraction contains highly enriched mitochondria.
• Fig.3-3A : equipment for isoelectric focusing
• Fig. 3-3B : equipment for SDS-PAGE
• Fig. 3-4A: the first step of database search for proteins . Example of the parameters used for the database search with the Mascot search engine. The parameters such as taxo­nomy, allowed missed cleavages, variable modifications, and peptide mass tolerance were restricted as shown. Only the mono­isotopic peptide masses were considered in this search.
• Fig. 3-4B: the second step of database search for proteins . The first protein is usually the best fit. The full name of the candidate protein together with its gi-number, the theoretical molecular weight, the probability based Mowse-score and the number of matched peptides are all listed. Normally, there are seve­ral peptides or proteins listed in one suggestion. They generally are various frag­ments of the same protein.
• Fig . 3-4C : The third step of the database search . The detailed view of a certain protein, e.g. the ATP synthase beta-chain, includes information on the protein and on the sequence of the protein covered by the peptide mass fingerprint (highlighted in red). The full information of the protein can be accessed from the NCBI database, by clicking on the accession number (gi number) of the protein.
• Fig. 4-2: Electron microscopic picture of a lymphoblastoid cell . Each cell contains about 15-20 mitochondria.
• Fig. 4-1: This summary describes the procedures and the results of this study step by step. The work started from the establishment of a lymphoblastoid cell line. The electron microscopic photographs showed that each lymphocyte contains about 15-20 mitochondria. The mitochondria were isolated after homogenisation of the lymphocytes followed by density gradient centrifugation. The three fractions were examined by electron microscopy. Only the third fraction was enriched of mitochondria. This fraction was used in subsequent experiments. The mitochondrial proteins were separated by 2D-electrophoresis and stained with Coomassie brilliant blue. The various protein-spots were cut out and were digested with trypsin. A peptide mass fingerprint or a peptide ladder were then generated with MALDI-TOF or MALDI-QTOF respectively. After database searching, the identified proteins were marked on the 2D-electrophoresis gel in order to obtain the final reference map.
• Fig. 4-3: Comparison of the 2D-electrophoresis maps of lymphoblastoid cells and of isolated mitochondria . Sub-cellular fractionation can intensify low abundant proteins and let them become visible. (A) depicts the map of lymphoblastoid cells, (B) depicts the map of the mitochondrial subfaction. The red arrows point to some intensified protein spots on the mitochondrial map. The green arrows point to the corresponding spots in the lymphoblastoid map.
• Fig. 4-4: Analysis of the protein concentration with the BCA protein assay. The result has to be multiplied by ten (highlighted with red), since the sample had been diluted 10 times before the measurement.
• Fig. 4-5A-B : pH-gradient of 40 cm IEF gels . The pH-measurement was performed as described in section 3.5.4.3. The results of the empty control gel are depicted in (A). The measured spots tend to lie on a regression line (y = -0.1088x+8.8965, R2 = 0.939 (p<0.0001)). The gradient is linear between the pH-values of 5-9. The effect of the protein sample on the pH-gradient is shown in (B). Although the pH-gradient of the protein containing gel fluctuated more than that of the empty gel, the linear regression lines are similar (y= -0.098x+8.85, R2 = 0.922 (p<0.0001)).
• Fig.4-6: These four corresponding gel-sections demonstrate the reproducibility of 2D-electrophoresis . All four gels were stained with silver.
• Fig. 4-7: These two gel-sections demonstrate the difference in sensitivity between the silver (A) and the Coomassie G-250-stain (B). Both gels have been run under the same conditions. A total of 525 µg mitochondrial protein were loaded on gel B. In contrast, only 47 µg protein were loaded on gel A. Moreover, it achieved a higher resolution than gel B.
• Fig. 4-8: The effect of repeated freeze-thaw cycles on sample quality : Comparison of the two gels that have been prepared from frozen stocks. Gel A used the sample that was run on the 2D-gel after the first time thawing. Gel B used the sample that was run on the 2D-gel after an additional round of freezing and thawing. Although the amount of loaded sample of gel B was 2 µl more than that of gel A, many spots on gel B are much fainter than the corresponding ones on gel A.
• Fig. 4-9: The mass spectrum of spot 3. A total of 60 peptides were detected by MALDI-TOF analysis. Using the Mascot search engine, 28 peptides labelled with a green dot ( ) could be matched to the theoretical trypsin digest of the protein (ATP-synthese beta-chain, mitochondrial precursor ). The parameters for this search were one missed cleavage, possible methionine oxidation and 0.1 Da mass tolerance. All methionine containing peptides and their oxidized derivatives are grouped with the red M (M ). The presence of double-peaks separated by 16 Da confirms the presence of methionine residues in the respective peptide fragment. One fragment resulting form trypsin self-digestion is marked with pink color (trypsin ). When less stringent criteria were used, some more peptides could be matched. If the mass tolerance is increased to 0.5 Da, additional fragments can be matched ( ). If the number of possible missed cleavages is increased to four, an additional large fragment can be matched ( ).
• Fig. 4-10A: The mass spectrum of spot 30 measured with MALDI-TOF mass spectrometry. Only 6 peptides were detected including 3 peptides of the self-digested trypsin.
• Fig. 4-10B: The mass spectrum of spot 30 after desalting with nano-scale reversed-phase chromatography. A total of 22 peptides was now detected by MALDI-TOF mass spectrometry. One isolated peptide was selected out for further MALDI-QTOF measurement (highlighted with a red arrow). Peptide ladder sequencing had to be performed because this spectrum was still not good enough to identify the protein.
• Fig. 4-10C : The mass spectrum of the 1168.67 Da peptide of spot 30 using MALDI-QTOF tandem mass spectrometry. These fragments are used to generate the protein sequence tag by GPMAW32-software on the internet (see following figure).
• Fig. 4-10D: The sequence result of the isolated 1168.67 Da peptide of spot 30. The peptide fragment ladders were obtained by the MALDI-QTOF mass spectrometry. This sequence tag was later used for database searching in order to identify the protein.
• Fig. 4-11 : Strategy of protein identification.
• Fig. 4-13 : Classification of the identified proteins according to their location. The mitochondrial group (M) includes those proteins that are definitively mitochondrial but for which no further information exists on their exact location.
• Fig. 4-12: The reference map of the mitochondrial proteome from human lymphoblastoid cells . All the measured spots are highlighted (red : mitochondrial proteins; green : other cytoplasmic proteins; yellow : proteins which locations remain unknown). The corresponding spot numbers are shown at the spots. The MW and pI ranges of the whole gel are shown at the left and above the gel.
• Fig. 4-14: Identified mitochondrial membrane proteins . Among the 74 identified mitochondrial proteins, only four carry transmembrane domains. One spot (#75) belongs to complex I of the respiratory chain, other two spots (#13 and #90) function in the citrate acid cycle and the fourth spot (#134) is an iron transport protein.
• Fig. 4-15: multiple spots proteins. A total of 17 proteins on the reference map could be detected in more than one spot. Most of the multiple spot proteins are probably isoforms of the same proteins. However, spots 5 and 31 (complex IV, subunit Va) correspond to the same gene but show a marked difference in pI and MW. Similar to that, spot 114 that was identified by sequence information, corresponds to the same gene as spots 11 and 12 (actin-beta). Spot 26, which originally corresponded to a unknown protein, was finally identified to be a short isoform of actin-beta.
• Fig. 4-16 : Comparison of the theoretical and experimental pI and MW of the identified proteins. The left regression diagram depicts the relation of the pI values between the measured values (m) and the theoretical calculations (S). Similar to that, the right regression diagram depicts the relation between the two different MW values. The linear regression formulas and the lateral dispersion (R2) are depicted below the diagrams.
• Fig. 5-1 : Comparison of my results to Rabilloud‘s and Fountoulakis‘ mitochondrial proteome databases. Compared to the 95 proteins in my database, the database of Rabilloud contains 68 proteins in total and the other database contains 185 different proteins. The shared proteins between the different databases are depicted in the overlaying parts.
• Fig. 4-12#1 : Section 1 of mitochondrial the proteome map
• Fig. 4-12#2 : Section 2 of the mitochondrial proteome map
• Fig. 4-12#3 : Section 3 of the mitochondrial proteome map
• Fig. 4-12#4 : Section 4 of the mitochondrial proteome map
• Fig. 4-12#5 : Section 5 of the mitochondrial proteome map
• Fig. 4-12#6 : Section 6 of the mitochondrial proteome map
• Fig. 4-12#7 : Section 7 of the mitochondrial proteome map
• Fig. 4-12#8 : Section 8 of the mitochondrial proteome map
• Fig. 4-12#9 : Section 9 of the mitochondrial proteome map
• Fig. 4-12#10 : Section 10 of the mitochondrial proteome map
• Fig. 4-12#11 : Section 11 of the mitochondrial proteome map
• Fig. 4-12#12 : Section 12 of the mitochondrial proteome map