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The impact of high-resolution mass spectrometry in shotgun proteomics : two case studies

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Presentation Outline. Proteomics : an introduction. Case study 1 : N. vitripennisvenomics. Biological objectives. Experimental set-up. Results. Outline. PhD. defense ... – PowerPoint PPT presentation

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Title: The impact of high-resolution mass spectrometry in shotgun proteomics : two case studies


1
(No Transcript)
2
The impact of high-resolution mass spectrometry
in shotgun proteomics two case studies
  • Maarten Aerts
  • Promotor Prof. Dr. B. Devreese
  • Co-Promotor Prof. Dr. Em. J. Van Beeumen

PhD. defense Maarten Aerts, May 6th 2010
3
Presentation Outline
  • Proteomics an introduction
  • Definitions
  • Mass Spectrometry
  • Protein/Peptide Identification
  • Impact of High-Resolution MS
  • Shotgun Approach

Outline
PhD. defense Maarten Aerts, May 6th 2010
4
Presentation Outline
  • Proteomics an introduction
  • Case study 1 N. vitripennis venomics
  • Biological objectives
  • Experimental set-up
  • Results

Outline
PhD. defense Maarten Aerts, May 6th 2010
5
Presentation Outline
  • Proteomics an introduction
  • Case study 1 N. vitripennis venomics
  • Case study 2 Comparative proteomics on
    ciprofloxacin resistance
  • Comparative proteomics
  • Medicinal objectives
  • Experimental set-up
  • Results

PhD. defense Maarten Aerts, 6th may 2010
Outline
6
Presentation Outline
  • Proteomics an introduction
  • Case study 1 N. vitripennis venomics
  • Case study 2 Comparative proteomics on
    ciprofloxacin resistance
  • Conclusions and future perspectives

Outline
PhD. defense Maarten Aerts, May 6th 2010
7
Proteomics Introduction
  • Central Dogma
  • DNA
  • mRNA
  • Protein

P
Duplication
Transcription
Translation
Introduction
PhD. defense Maarten Aerts, May 6th 2010
8
Proteomics Introduction
  • Proteins
  • - amino acid polymer ? structure and function
  • - define a cells phenotype
  • - molecular active molecules
  • Structural proteins
  • Transport proteins
  • Enzymes

Introduction
PhD. defense Maarten Aerts, May 6th 2010
9
Proteomics Introduction
  • Proteins
  • Proteome total complement of all proteins
    present in
  • a particular cell
  • at a given time
  • in a given environment
  • ? Dynamic alteration due to intra- and
    extracellular signals
  • Genome total complement of all the genes
    present in an organism
  • ? Static identical in all cells
  • Gene expression (mRNA or proteins) Gene
    function prediction

Introduction
PhD. defense Maarten Aerts, May 6th 2010
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Proteomics Introduction
Introduction
PhD. defense Maarten Aerts, May 6th 2010
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Proteomics Introduction
micro array fast and complete
?
slow and incomplete coverage
Introduction
PhD. defense Maarten Aerts, May 6th 2010
12
Proteomics Introduction
  • Central Dogma
  • DNA
  • mRNA
  • Protein

R
P
Duplication
Transcription
R
PTM Localization
R
Translation
Introduction
PhD. defense Maarten Aerts, May 6th 2010
13
Proteomics Introduction
micro array fast and complete
slow and incomplete coverage
Introduction
PhD. defense Maarten Aerts, May 6th 2010
14
Proteomics mass spectrometry
  • High-throughput analyses on proteomes,
    sub-proteomes or protein complexes based
    on mass spectrometry to identify, quantify
    and/or characterize proteins.
  • analytical technique to determine the molecular
    weight of molecules, including peptides or
    proteins

Introduction
PhD. defense Maarten Aerts, May 6th 2010
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Proteomics mass spectrometry
PhD. defense Maarten Aerts, May 6th 2010
Introduction
16
Proteomics mass spectrometry
ESI MALDI
Ionization
PhD. defense Maarten Aerts, May 6th 2010
Introduction
17
Proteomics mass spectrometry
ESI MALDI
Ionization
Quadrupole Ion Trap TOF FT-ICR Orbitrap Ion
Mobility
Mass analyzer
Detector
PhD. defense Maarten Aerts, May 6th 2010
Introduction
18
Proteomics mass spectrometry
ESI MALDI
Ionization
Quadrupole Ion Trap TOF FT-ICR Orbitrap Ion
Mobility
Mass analyzer
Detector
PhD. defense Maarten Aerts, May 6th 2010
Introduction
19
Proteomics mass spectrometry
PhD. defense Maarten Aerts, May 6th 2010
Introduction
20
Proteomics Protein/Peptide Identification
Trypsin K/R
MS
Unique Peptide Mass Fingerprint
Protein database ? prediction PMF
Introduction
PhD. defense Maarten Aerts, May 6th 2010
21
Proteomics Protein/Peptide Identification
Trypsin K/L
MS
Unique Peptide Fragment Fingerprint
MS/MS
Unique Peptide Mass Fingerprint
Introduction
PhD. defense Maarten Aerts, May 6th 2010
22
Proteomics Protein/Peptide Identification
ESI MALDI
Ionization
Quadrupole Ion Trap TOF FT-ICR Orbitrap Ion
Mobility
Mass analyzer
Tandem MS
CID ECD ETD
Detector
Introduction
PhD. defense Maarten Aerts, May 6th 2010
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Proteomics Protein/Peptide Identification
  • PMF
  • Peptide Mass Fingerprint
  • Single protein
  • Database searching
  • ? Unique MS fingerprint
  • PFF
  • Peptide Fragment Fingerprint
  • Peptide selection
  • Database searching
  • ? Amino acid sequence (MS/MS)

Introduction
PhD. defense Maarten Aerts, May 6th 2010
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High-Resolution Mass Spectrometry
  • High-throughput analyses on proteomes,
    sub-proteomes or protein complexes based
    on mass spectrometry to identify, quantify
    and/or characterize proteins.
  • analytical technique to determine the molecular
    masses of molecules, including peptides or
    proteins
  • High-Resolution MS

Introduction
PhD. defense Maarten Aerts, May 6th 2010
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High-Resolution Mass Spectrometry
  • Rs peak width at half peak intensity
  • ? Sharp peaks
  • Peak capacity
  • Peak detection
  • Peak selection
  • Charge state determination
  • Mass accuracy

Introduction
PhD. defense Maarten Aerts, May 6th 2010
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High-Resolution Mass Spectrometry
  • Rs peak width at half peak intensity
  • ? Sharp peaks
  • Peak capacity isobaric peptides
  • Peak detection
  • Peak selection
  • Charge state determination
  • Mass accuracy

Introduction
PhD. defense Maarten Aerts, May 6th 2010
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High-Resolution Mass Spectrometry
  • Rs peak width at half peak intensity
  • ? Sharp peaks
  • Peak capacity
  • Peak detection
  • Peak selection
  • Charge state determination
  • Mass accuracy

Introduction
PhD. defense Maarten Aerts, May 6th 2010
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High-Resolution Mass Spectrometry
  • Rs peak width at half peak intensity
  • ? Sharp peaks
  • Peak capacity
  • Peak detection
  • Peak selection
  • Charge state determination
  • z m
  • Mass accuracy

Introduction
PhD. defense Maarten Aerts, May 6th 2010
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High-Resolution Mass Spectrometry
  • Rs peak width at half peak intensity
  • ? Sharp peaks
  • Peak capacity
  • Peak detection
  • Peak selection
  • Charge state determination
  • Mass accuracy database searching

Introduction
PhD. defense Maarten Aerts, May 6th 2010
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High-Resolution Mass Spectrometry
  • Rs peak width at half peak intensity
  • ? Sharp peaks
  • Peak capacity
  • Peak detection
  • Peak selection
  • Charge state determination
  • Mass accuracy database searching
  • ? peptide identification
  • ? peptide quantification

Introduction
PhD. defense Maarten Aerts, May 6th 2010
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High-Resolution Mass Spectrometry
  • Rs peak width at half peak intensity
  • FT-ICR Fourier Transformation Ion Cyclotron
    Resonance

Magnetic field, B
Cyclotron frequency,
Introduction
PhD. defense Maarten Aerts, May 6th 2010
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High-Resolution Mass Spectrometry
Introduction
PhD. defense Maarten Aerts, May 6th 2010
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High-Resolution Mass Spectrometry
  • Rs peak width at half peak intensity
  • FT-ICR Fourier Transformation Ion Cyclotron
    Resonance

Magnetic field, B
Cyclotron frequency,
m/z
Introduction
PhD. defense Maarten Aerts, May 6th 2010
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High-Resolution Mass Spectrometry
  • Rs peak width at half peak intensity
  • FT-ICR Fourier Transformation Ion Cyclotron
    Resonance

Magnetic field, B
Cyclotron frequency,
m/z
Multiple m/z complex waveform
FT
Introduction
PhD. defense Maarten Aerts, May 6th 2010
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High-Resolution Mass Spectrometry
  • Rs peak width at half peak intensity
  • FT-ICR Fourier Transformation Ion Cyclotron
    Resonance

Magnetic field, B
Cyclotron frequency,
Multiple m/z complex waveform
Slow
Introduction
PhD. defense Maarten Aerts, May 6th 2010
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High-Resolution Mass Spectrometry
  • Rs peak width at half peak intensity
  • FT-ICR Fourier Transformation Ion Cyclotron
    Resonance
  • LTQ-FTUltra MS

FT-MS
LTQ
PhD. defense Maarten Aerts, May 6th 2010
Introduction
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High-Resolution Mass Spectrometry
  • Rs peak width at half peak intensity
  • FT-ICR Fourier Transformation Ion Cyclotron
    Resonance
  • LTQ-FTUltra MS

PhD. defense Maarten Aerts, May 6th 2010
Introduction
38
High-Resolution Mass Spectrometry
  • Rs peak width at half peak intensity
  • FT-ICR Fourier Transformation Ion Cyclotron
    Resonance
  • LTQ-FTUltra MS
  • Data-Dependent Acquisition
  • ? Ideal LC-MS platform
  • high quality MS
  • high quantity MS/MS

PhD. defense Maarten Aerts, May 6th 2010
Introduction
39
Protein/Peptide separation
  • High-throughput analyses on proteomes,
    sub-proteomes or protein complexes based
    on mass spectrometry to identify, quantify
    and/or characterize proteins.
  • analytical technique to determine the molecular
    masses of molecules, including peptides or
    proteins
  • High-Resolution MS
  • Sample complexity ? fractionation strategies

PhD. defense Maarten Aerts, May 6th 2010
Introduction
40
pI / MW
41
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n x Dim-LC
  • - SEC
  • SCX
  • RP
  • - CF

MS and MS/MS on intact protein (Top-Down)
Digest on single protein
MS and MS/MS
PMF or PFF
43
n x Dim-LC
Limitations Dynamic range low abundant
Range physicochemical properties pI, MW and
hydrophobicity
  • - SEC
  • SCX
  • RP
  • - CF

MS and MS/MS on intact protein (Top-Down)
Digest on single protein
MS and MS/MS
PMF or PFF
44
Protein mixture
n x Dim-LC
GeLC
2D-PAGE
  • - SEC
  • SCX
  • RP
  • - CF

Digest on gel band containing few proteins
Digest on single spot containing one protein
MS and MS/MS on intact protein (Top-Down)
Digest on single protein
MS and MS/MS
LC-MS/MS
MS and MS/MS
PMF or PFF
PFF
PMF or PFF
45
Protein mixture
Shotgun proteomics
GeLC
n x Dim-LC
2D-PAGE
  • - SEC
  • SCX
  • RP
  • - CF

Digest on complete protein mixture
Enrichment of targeted peptides
Digest on gel band containing few proteins
Digest on single spot containing one protein
MS and MS/MS on intact protein (Top-Down)
Digest on single protein
  • 2D LC-MS/MS
  • SCX
  • RP

LC-MS/MS
MS and MS/MS
MS and MS/MS
PFF
PMF or PFF
PMF or PFF
PFF
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Protein mixture
  • Shotgun proteomics
  • Tryptic digest
  • ? complexity
  • ? multidimensional LC
  • ?Peptide sequencing MS/MS
  • Analyzable peptides represent proteins
  • ? Proteome coverage

Shotgun proteomics
GeLC
Digest on complete protein mixture
Enrichment of targeted peptides
Digest on gel band containing few proteins
  • 2D LC-MS/MS
  • SCX
  • RP

LC-MS/MS
PFF
PFF
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Shotgun Proteomics
  • Shotgun proteomics
  • Tryptic digest
  • ? complexity
  • ? Bottom-up proteomics
  • Analyzable peptides represent proteins
  • Peptide sequencing MS/MS
  • ? Sensitivity
  • ? Dynamic range
  • Top-down proteomics
  • Intact proteins
  • Limited proteome coverage
  • Protein sequencing MS/MS

Introduction
PhD. defense Maarten Aerts, May 6th 2010
48
Shotgun Proteomics
  • LC-MS platform
  • - Multi-dimensional LC
  • - High-Resolution MS
  • - MS/MS database searching
  • Application
  • Venom proteins of Nasonia vitripennis (parasitoid
    wasp)
  • Drug resistance mechanism on mouse macrophages

Introduction
PhD. defense Maarten Aerts, May 6th 2010
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Nasonia Venomics Introduction
  • Nasonia vitripennis
  • Ectoparasitoid wasp

Nasonia Venomics
PhD. defense Maarten Aerts, May 6th 2010
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Nasonia Venomics Introduction
  • Nasonia vitripennis
  • Ectoparasitoid wasp

Nasonia Venomics
PhD. defense Maarten Aerts, May 6th 2010
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Nasonia Venomics Introduction
  • Nasonia vitripennis
  • Ectoparasitoid wasp

Nasonia Venomics
PhD. defense Maarten Aerts, May 6th 2010
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Nasonia Venomics Introduction
  • Nasonia vitripennis
  • Ectoparasitoid wasp

Nasonia Venomics
PhD. defense Maarten Aerts, May 6th 2010
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Nasonia Venomics Introduction
  • Nasonia vitripennis
  • Ectoparasitoid wasp

Venom proteins - Host behavior - Growth
alteration - Immune suppression - Nutrient
metabolism
Nasonia Venomics
PhD. defense Maarten Aerts, May 6th 2010
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Nasonia Venomics Introduction
  • Nasonia vitripennis
  • Ectoparasitoid wasp
  • 50 years scientific history
  • Biological model organism parasitoids
  • - controller of insect populations
  • crop pests
  • disease vectors
  • - commercialized

Nasonia Venomics
PhD. defense Maarten Aerts, May 6th 2010
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Nasonia Venomics Introduction
  • Nasonia vitripennis
  • Ectoparasitoid wasp
  • 50 years scientific history
  • Biological model organism parasitoids
  • Genetic model organism
  • - Ease of breeding
  • - Haplodiploidy (recessive vs. dominance)
  • - Inter-fertility N. giraulti, N. longcornis
  • (Complex trait analyses)
  • ? Genome sequence

Nasonia Venomics
PhD. defense Maarten Aerts, May 6th 2010
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Nasonia Venomics Introduction
  • Genome sequence
  • Characterization of Venom
  • - Venom experience (bee)
  • - Function during reproduction
  • - Host behavior
  • - Growth alteration
  • - Immune suppression
  • - Nutrient metabolism
  • - Bioinformatic approach venom protein
    prediction
  • ? candidate gene list
  • ? confirmation at mRNA
  • - Proteomic approach

Bee venom defensive
Nasonia Venomics
PhD. defense Maarten Aerts, May 6th 2010
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Nasonia Venomics Proteomic analysis
10 venom reservoirs
Trypsin
Shotgun proteomic approach via LC-MS platform -
2D-LC first dimension SCX (charge) second
dimension RP (hydrophobicity)
Nasonia Venomics
PhD. defense Maarten Aerts, May 6th 2010
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Nasonia Venomics Proteomic analysis
10 venom reservoirs
Trypsin
Shotgun proteomic approach via LC-MS platform -
2D-LC first dimension SCX (charge) second
dimension RP (hydrophobicity) - MALDI
TOF-TOF 29 peptides ?14 proteins - ESI
FT-MS 258 peptides ?76 proteins
Nasonia Venomics
PhD. defense Maarten Aerts, May 6th 2010
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Nasonia Venomics Results
  • 76 proteins 61 secretion signal
  • limited overlap with bioinformatic approach
  • ? complexity
  • ? venom reservoir ? venom gland
  • ? 23 unknown function

Nasonia Venomics
PhD. defense Maarten Aerts, May 6th 2010
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Nasonia Venomics Future directions
  • First identification of venom proteins
  • Characterization of the molecular mechanism
    behind venom activity
  • efficiency as pest controller agriculture and
    human health
  • ? pharmaceutical applications
  • ? transgenic plants or viruses

Nasonia Venomics
PhD. defense Maarten Aerts, May 6th 2010
61
Case Study 2 Ciprofloxacin resistance
PhD. defense Maarten Aerts, May 6th 2010
Ciprofloxacin resistance
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Ciprofloxacin resistance
  • Intracellular accumulating antibiotic
  • ? intracellular infections e.g. Listeria
    monocytogenes

PhD. defense Maarten Aerts, May 6th 2010
Ciprofloxacin resistance
63
Ciprofloxacin resistance
  • Intracellular accumulating antibiotic
  • ? intracellular infections e.g. Listeria
    monocytogenes

PhD. defense Maarten Aerts, May 6th 2010
Ciprofloxacin resistance
64
Ciprofloxacin resistance
  • Intracellular accumulating antibiotic
  • ? intracellular infections e.g. Listeria
    monocytogenes

PhD. defense Maarten Aerts, May 6th 2010
Ciprofloxacin resistance
65
Ciprofloxacin resistance
  • Intracellular accumulating antibiotic
  • Long-term exposure to ciprofloxacin ? eukaryotic
    resistance

PhD. defense Maarten Aerts, May 6th 2010
Ciprofloxacin resistance
66
Ciprofloxacin resistance
  • Intracellular accumulating antibiotic
  • Long-term exposure to ciprofloxacin ? eukaryotic
    resistance

PhD. defense Maarten Aerts, May 6th 2010
Ciprofloxacin resistance
67
Ciprofloxacin resistance
  • Intracellular accumulating antibiotic
  • Long-term exposure to ciprofloxacin ? eukaryotic
    resistance

PhD. defense Maarten Aerts, May 6th 2010
68
Ciprofloxacin resistance
  • Intracellular accumulating antibiotic
  • Long-term exposure to ciprofloxacin ? eukaryotic
    resistance

PhD. defense Maarten Aerts, May 6th 2010
69
Ciprofloxacin resistance
  • Intracellular accumulating antibiotic
  • Long-term exposure to ciprofloxacin ? eukaryotic
    resistance

PhD. defense Maarten Aerts, May 6th 2010
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Ciprofloxacin resistance
  • Intracellular accumulating antibiotic
  • Long-term exposure to ciprofloxacin ? eukaryotic
    resistance

PhD. defense Maarten Aerts, May 6th 2010
Ciprofloxacin resistance
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Ciprofloxacin resistance
  • Intracellular accumulating antibiotic
  • Long-term exposure to ciprofloxacin ? eukaryotic
    resistance

PhD. defense Maarten Aerts, May 6th 2010
72
Ciprofloxacin resistance
  • Intracellular accumulating antibiotic
  • Long-term exposure to ciprofloxacin ? eukaryotic
    resistance

PhD. defense Maarten Aerts, May 6th 2010
73
Ciprofloxacin resistance
  • Intracellular accumulating antibiotic
  • Long-term exposure to ciprofloxacin ? eukaryotic
    resistance

PhD. defense Maarten Aerts, May 6th 2010
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Ciprofloxacin resistance
  • Intracellular accumulating antibiotic
  • Long-term exposure to ciprofloxacin ? eukaryotic
    resistance
  • ? life-threatening infections
  • ? ATP-driven
  • ? Inhibitors
  • ? Multidrug Resistance Proteins
  • Comparative proteomic analysis on membrane
    proteomes

PhD. defense Maarten Aerts, May 6th 2010
Ciprofloxacin resistance
75
Comparative proteomics
PhD. defense Maarten Aerts, May 6th 2010
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Comparative proteomics
WT Heavy labeled
CIP Light labeled
Tryptic digest
LC-MS
PhD. defense Maarten Aerts, May 6th 2010
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Comparative proteomics
WT Heavy labeled
CIP Light labeled
Tryptic digest
LC-MS
PhD. defense Maarten Aerts, May 6th 2010
78
Comparative proteomics
PhD. defense Maarten Aerts, May 6th 2010
Ciprofloxacin resistance
79
Comparative proteomics
PhD. defense Maarten Aerts, May 6th 2010
Ciprofloxacin resistance
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Results
  • LC-MS (FT-MS)
  • 900 protein identified and (relatively)
    quantified
  • ?136 proteins with significant
  • abundance difference between
  • WT and CIP macrophages
  • MRP4 (ABCC4)
  • - Multidrug Resistance protein
  • - ATP

PhD. defense Maarten Aerts, May 6th 2010
Ciprofloxacin resistance
81
Discussion
MRP4
Confirmed - RT-PCR (mRNA) - Western blot
(protein)
PhD. defense Maarten Aerts, May 6th 2010
Ciprofloxacin resistance
82
Discussion
  • MRP4 (ABCC4)
  • p58IPK Negative regulator of UPR evoked by ER
    stress
  • ? CIP resistance Stress resistance

PhD. defense Maarten Aerts, May 6th 2010
Ciprofloxacin resistance
83
Discussion
  • MRP4 (ABCC4)
  • p58IPK Negative regulator of UPR evoked by ER
    stress
  • ? CIP resistance Stress resistance

PhD. defense Maarten Aerts, May 6th 2010
Ciprofloxacin resistance
84
Discussion
  • MRP4 (ABCC4)
  • p58IPK Negative regulator of UPR evoked by ER
    stress
  • ? CIP resistance Stress resistance
  • ? MRP4 over-expression ? ER stress
  • ? No other UPR proteins are differentially
    expressed
  • ! Genome organization

PhD. defense Maarten Aerts, May 6th 2010
Ciprofloxacin resistance
85
Discussion
Chr. 14
mrp4
p58IPK
? Gene duplication
PhD. defense Maarten Aerts, May 6th 2010
Ciprofloxacin resistance
86
Discussion
  • MRP4 (ABCC4)
  • p58IPK Negative regulator of UPR evoked by ER
    stress
  • Remaining proteins
  • ? related to drug resistance ?
  • ? secondary effects ?
  • MRP4 cAMP, Leukotriens, etc.
  • p58IPK regulator of Transcription Factors

PhD. defense Maarten Aerts, May 6th 2010
Ciprofloxacin resistance
87
Conclusion
  • Identified ciprofloxacin efflux pump MRP4
  • mode of induction gene duplication
  • A model for in vivo drug resistance mechanism
  • - MRP family broad substrate specificity
  • ? Antibiotics
  • ? Anti-viral drug
  • ? Chemotherapeutics
  • - Secondary effects
  • ? Stress resistance
  • ? cell motility and adherence
  • ? creating an environment for new resistance
    mechanism

PhD. defense Maarten Aerts, May 6th 2010
Ciprofloxacin resistance
88
Conclusion
PhD. defense Maarten Aerts, May 6th 2010
Ciprofloxacin resistance
89
Conclusion
PhD. defense Maarten Aerts, May 6th 2010
Ciprofloxacin resistance
90
General Conclusions
PhD. defense Maarten Aerts, May 6th 2010
Conclusions
91
General Conclusions
  • Shotgun proteomics relevant biological and
    medicinal problems
  • ? LC-MS platform
  • 2D-LC or GeLC
  • Database searching
  • Automated statistical validation software
  • High-Resolution MS FT-ICR
  • ? mass accuracy
  • ? peptide coverage
  • ? protein coverage
  • ? peak detection
  • ? quantification

PhD. defense Maarten Aerts, May 6th 2010
Conclusions
92
General Conclusions
  • Shotgun proteomics relevant biological and
    medicinal problems
  • ? LC-MS platform
  • ? Nasonia venomics new venom proteins
  • ? Ciprofloxacin resistance mechanism MRP4,
    gene duplication
  • ? Start of new discovery phase

PhD. defense Maarten Aerts, May 6th 2010
Conclusions
93
Future perspectives
  • Holy grail sensitivity Complete
    proteome coverage
  • LC-MS platforms
  • - Mass spectrometry
  • mass analyzers
  • ionization
  • - Peptide fractionation
  • chip-based LC
  • - Data analysis
  • MS/MS homology Estimation of FPR
  • MS/MS independent identification

PhD. defense Maarten Aerts, May 6th 2010
Future perspectives
94
Acknowledgement
  • Promotor Prof. Dr. B. Devreese
  • Co-Promotor Prof. Dr. J. Van Beeumen
  • Friends and Family
  • L-ProBE colleagues

Unité de Pharamcologie Cellulaire et Moléculaire
(UCL) prof. Dr. P. Tulkens prof. Dr. F. Van
Bambeke Nancy Caceres Beatrice Marquez
Laboratory of Zoophysiology prof. Dr. D. de
Graaf Marleen Brunain Ellen Danneels Ellen
Formesyn
PhD. defense Maarten Aerts, May 6th 2010
Acknowlegment
95
?
PhD. defense Maarten Aerts, May 6th 2010
Questions
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