The impact of high-resolution mass spectrometry in shotgun proteomics : two case studies Maarten...
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- 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 6 th 2010
- Slide 3
- Presentation Outline Proteomics : an introduction Definitions
Mass Spectrometry Protein/Peptide Identification Impact of
High-Resolution MS Shotgun Approach OutlinePhD. defense : Maarten
Aerts, May 6 th 2010
- Slide 4
- Presentation Outline Proteomics : an introduction Case study 1
: N. vitripennis venomics Biological objectives Experimental set-up
Results OutlinePhD. defense : Maarten Aerts, May 6 th 2010
- Slide 5
- PhD. defense : Maarten Aerts, 6 th may 2010 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 Outline
- Slide 6
- Presentation Outline Proteomics : an introduction Case study 1
: N. vitripennis venomics Case study 2 : Comparative proteomics on
ciprofloxacin resistance Conclusions and future perspectives
OutlinePhD. defense : Maarten Aerts, May 6 th 2010
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- Central Dogma DNA mRNA Protein Proteomics : Introduction
Introduction P Transcription Translation Duplication PhD. defense :
Maarten Aerts, May 6 th 2010
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- Proteins : - amino acid polymer structure and function - define
a cells phenotype - molecular active molecules Structural proteins
Transport proteins Enzymes Proteomics : Introduction
IntroductionPhD. defense : Maarten Aerts, May 6 th 2010
- Slide 9
- 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 Proteomics : Introduction IntroductionPhD.
defense : Maarten Aerts, May 6 th 2010
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- Proteomics : Introduction Introduction Genomics Proteomics
Transcriptomics Gene function Gene prediction Gene function PhD.
defense : Maarten Aerts, May 6 th 2010
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- Proteomics : Introduction Introduction Genomics Proteomics
Transcriptomics Gene function Gene prediction Gene function micro
array : fast and complete slow and incomplete coverage ? PhD.
defense : Maarten Aerts, May 6 th 2010
- Slide 12
- Central Dogma DNA mRNA Protein Proteomics : Introduction
Introduction P Transcription Translation Duplication R R R PTM
Localization PhD. defense : Maarten Aerts, May 6 th 2010
- Slide 13
- Proteomics : Introduction Introduction Genomics Proteomics
Transcriptomics Gene function Gene prediction Gene function micro
array : fast and complete slow and incomplete coverage PhD. defense
: Maarten Aerts, May 6 th 2010
- Slide 14
- 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
Proteomics : mass spectrometry IntroductionPhD. defense : Maarten
Aerts, May 6 th 2010
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- Sample Peptides Proteins Proteomics : mass spectrometry PhD.
defense : Maarten Aerts, May 6 th 2010Introduction
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- Sample Peptides Proteins Ionization ESI MALDI Proteomics : mass
spectrometry PhD. defense : Maarten Aerts, May 6 th
2010Introduction
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- Sample Peptides Proteins Ionization ESI MALDI Detector Mass
analyzer Quadrupole Ion Trap TOF FT-ICR Orbitrap Ion Mobility
Proteomics : mass spectrometry PhD. defense : Maarten Aerts, May 6
th 2010Introduction
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- Sample Peptides Proteins Ionization ESI MALDI Detector Mass
analyzer Quadrupole Ion Trap TOF FT-ICR Orbitrap Ion Mobility
Proteomics : mass spectrometry PhD. defense : Maarten Aerts, May 6
th 2010Introduction
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- Proteomics : mass spectrometry PhD. defense : Maarten Aerts,
May 6 th 2010Introduction
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- Proteomics : Protein/Peptide Identification Trypsin : K/R m/z
MS Unique Peptide Mass Fingerprint Protein database prediction PMF
IntroductionPhD. defense : Maarten Aerts, May 6 th 2010
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- Proteomics : Protein/Peptide Identification Trypsin : K/L m/z
MS Unique Peptide Mass Fingerprint MS/MS m/z Unique Peptide
Fragment Fingerprint m/z IntroductionPhD. defense : Maarten Aerts,
May 6 th 2010
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- Sample Peptides Proteins Ionization ESI MALDI Detector Mass
analyzer Quadrupole Ion Trap TOF FT-ICR Orbitrap Ion Mobility
Tandem MS CID ECD ETD Proteomics : Protein/Peptide Identification
IntroductionPhD. defense : Maarten Aerts, May 6 th 2010
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- PMF Peptide Mass Fingerprint Single protein Database searching
Unique MS fingerprint PFF Peptide Fragment Fingerprint Peptide
selection Database searching Amino acid sequence (MS/MS) m/z
Proteomics : Protein/Peptide Identification IntroductionPhD.
defense : Maarten Aerts, May 6 th 2010
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- 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 High-Resolution Mass Spectrometry
IntroductionPhD. defense : Maarten Aerts, May 6 th 2010
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- Rs : peak width at half peak intensity Sharp peaks Peak
capacity Peak detection Peak selection Charge state determination
Mass accuracy High-Resolution Mass Spectrometry IntroductionPhD.
defense : Maarten Aerts, May 6 th 2010
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- Rs : peak width at half peak intensity Sharp peaks Peak
capacity : isobaric peptides Peak detection Peak selection Charge
state determination Mass accuracy High-Resolution Mass Spectrometry
IntroductionPhD. defense : Maarten Aerts, May 6 th 2010
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- Rs : peak width at half peak intensity Sharp peaks Peak
capacity Peak detection Peak selection Charge state determination
Mass accuracy High-Resolution Mass Spectrometry IntroductionPhD.
defense : Maarten Aerts, May 6 th 2010
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- Rs : peak width at half peak intensity Sharp peaks Peak
capacity Peak detection Peak selection Charge state determination :
zm Mass accuracy High-Resolution Mass Spectrometry IntroductionPhD.
defense : Maarten Aerts, May 6 th 2010
- Slide 29
- Rs : peak width at half peak intensity Sharp peaks Peak
capacity Peak detection Peak selection Charge state determination
Mass accuracy : database searching High-Resolution Mass
Spectrometry IntroductionPhD. defense : Maarten Aerts, May 6 th
2010
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- 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 High-Resolution Mass Spectrometry IntroductionPhD.
defense : Maarten Aerts, May 6 th 2010
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- Rs : peak width at half peak intensity FT-ICR : Fourier
Transformation Ion Cyclotron Resonance High-Resolution Mass
Spectrometry Cyclotron frequency, Magnetic field, B
IntroductionPhD. defense : Maarten Aerts, May 6 th 2010
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- High-Resolution Mass Spectrometry IntroductionPhD. defense :
Maarten Aerts, May 6 th 2010
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- Rs : peak width at half peak intensity FT-ICR : Fourier
Transformation Ion Cyclotron Resonance High-Resolution Mass
Spectrometry Cyclotron frequency, Magnetic field, B Introduction ~
m/z PhD. defense : Maarten Aerts, May 6 th 2010
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- Rs : peak width at half peak intensity FT-ICR : Fourier
Transformation Ion Cyclotron Resonance High-Resolution Mass
Spectrometry Cyclotron frequency, Magnetic field, B Multiple
m/zcomplex waveform FT m/z Introduction ~ m/z PhD. defense :
Maarten Aerts, May 6 th 2010
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- Rs : peak width at half peak intensity FT-ICR : Fourier
Transformation Ion Cyclotron Resonance High-Resolution Mass
Spectrometry Cyclotron frequency, Magnetic field, B Multiple
m/zcomplex waveform Slow IntroductionPhD. defense : Maarten Aerts,
May 6 th 2010
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- Rs : peak width at half peak intensity FT-ICR : Fourier
Transformation Ion Cyclotron Resonance LTQ-FTUltra MS
High-Resolution Mass Spectrometry LTQ FT-MS PhD. defense : Maarten
Aerts, May 6 th 2010Introduction
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- Rs : peak width at half peak intensity FT-ICR : Fourier
Transformation Ion Cyclotron Resonance LTQ-FTUltra MS
High-Resolution Mass Spectrometry PhD. defense : Maarten Aerts, May
6 th 2010Introduction
- Slide 38
- 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 High-Resolution Mass Spectrometry PhD. defense
: Maarten Aerts, May 6 th 2010Introduction
- Slide 39
- 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
Protein/Peptide separation PhD. defense : Maarten Aerts, May 6 th
2010Introduction
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- Protein mixture 2D-PAGE Digest on single spot containing one
protein MS and MS/MS PMF or PFF pI / MW
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- Protein mixture 2D-PAGE Digest on single spot containing one
protein MS and MS/MS PMF or PFF
- Slide 42
- Protein mixture 2D-PAGE Digest on single spot containing one
protein MS and MS/MS PMF or PFF n x Dim-LC - SEC - SCX - RP - CF
Digest on single protein MS and MS/MS on intact protein (Top-Down)
MS and MS/MS PMF or PFF
- Slide 43
- Protein mixture 2D-PAGE Digest on single spot containing one
protein MS and MS/MS PMF or PFF n x Dim-LC - SEC - SCX - RP - CF
Digest on single protein MS and MS/MS on intact protein (Top-Down)
MS and MS/MS PMF or PFF Limitations : Dynamic range : low abundant
Range physicochemical properties pI, MW and hydrophobicity
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- n x Dim-LC - SEC - SCX - RP - CF Digest on single protein MS
and MS/MS on intact protein (Top-Down) MS and MS/MS PMF or PFF GeLC
Digest on gel band containing few proteins LC-MS/MS PFF 2D-PAGE
Digest on single spot containing one protein MS and MS/MS PMF or
PFF Protein mixture
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- GeLC Digest on gel band containing few proteins LC-MS/MS PFF n
x Dim-LC - SEC - SCX - RP - CF Digest on single protein MS and
MS/MS on intact protein (Top-Down) MS and MS/MS PMF or PFF 2D-PAGE
Digest on single spot containing one protein MS and MS/MS PMF or
PFF Shotgun proteomics Digest on complete protein mixture
Enrichment of targeted peptides 2D LC-MS/MS - SCX - RP PFF
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- Protein mixture Shotgun proteomics Digest on complete protein
mixture Enrichment of targeted peptides 2D LC-MS/MS - SCX - RP PFF
Shotgun proteomics Tryptic digest : complexity multidimensional LC
Peptide sequencing : MS/MS Analyzable peptides represent proteins
Proteome coverage GeLC Digest on gel band containing few proteins
LC-MS/MS PFF
- Slide 47
- 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 IntroductionPhD. defense : Maarten
Aerts, May 6 th 2010
- Slide 48
- Shotgun Proteomics LC-MS platform : - Multi-dimensional LC -
High-Resolution MS - MS/MS database searching Application : 1.Venom
proteins of Nasonia vitripennis (parasitoid wasp) 2.Drug resistance
mechanism on mouse macrophages IntroductionPhD. defense : Maarten
Aerts, May 6 th 2010
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- Nasonia vitripennis Ectoparasitoid wasp Nasonia Venomics :
Introduction Nasonia VenomicsPhD. defense : Maarten Aerts, May 6 th
2010
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- Nasonia vitripennis Ectoparasitoid wasp Nasonia Venomics :
Introduction Nasonia VenomicsPhD. defense : Maarten Aerts, May 6 th
2010
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- Nasonia vitripennis Ectoparasitoid wasp Nasonia Venomics :
Introduction Nasonia VenomicsPhD. defense : Maarten Aerts, May 6 th
2010
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- Nasonia vitripennis Ectoparasitoid wasp Nasonia Venomics :
Introduction Nasonia VenomicsPhD. defense : Maarten Aerts, May 6 th
2010
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- Nasonia vitripennis Ectoparasitoid wasp Nasonia Venomics :
Introduction Nasonia Venomics Venom proteins : - Host behavior -
Growth alteration - Immune suppression - Nutrient metabolism PhD.
defense : Maarten Aerts, May 6 th 2010
- Slide 54
- Nasonia vitripennis Ectoparasitoid wasp 50 years scientific
history Biological model organism : parasitoids - controller of
insect populations crop pests disease vectors - commercialized
Nasonia Venomics : Introduction Nasonia VenomicsPhD. defense :
Maarten Aerts, May 6 th 2010
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- 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 : Introduction
Nasonia VenomicsPhD. defense : Maarten Aerts, May 6 th 2010
- Slide 56
- 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 Nasonia Venomics :
Introduction Nasonia Venomics Bee venom : defensive PhD. defense :
Maarten Aerts, May 6 th 2010
- Slide 57
- Nasonia Venomics : Proteomic analysis Nasonia Venomics 10 venom
reservoirs Trypsin Shotgun proteomic approach via LC-MS platform -
2D-LC : first dimension SCX (charge) second dimension RP
(hydrophobicity) PhD. defense : Maarten Aerts, May 6 th 2010
- Slide 58
- Nasonia Venomics : Proteomic analysis Nasonia Venomics 10 venom
reservoirs Trypsin Shotgun proteomic approach via LC-MS platform -
2D-LC : first dimension SCX (charge) second dimension RP
(hydrophobicity) - MALDI TOF-TOF29 peptides 14 proteins - ESI
FT-MS258 peptides 76 proteins PhD. defense : Maarten Aerts, May 6
th 2010
- Slide 59
- 76 proteins :61 secretion signal limited overlap with
bioinformatic approach complexity venom reservoir venom gland 23
unknown function Nasonia Venomics : Results Nasonia VenomicsPhD.
defense : Maarten Aerts, May 6 th 2010
- Slide 60
- 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 : Future
directions Nasonia VenomicsPhD. defense : Maarten Aerts, May 6 th
2010
- Slide 61
- Case Study 2 : Ciprofloxacin resistance PhD. defense : Maarten
Aerts, May 6 th 2010Ciprofloxacin resistance
- Slide 62
- Intracellular accumulating antibiotic intracellular infections
e.g. Listeria monocytogenes Ciprofloxacin resistance PhD. defense :
Maarten Aerts, May 6 th 2010Ciprofloxacin resistance
- Slide 63
- Intracellular accumulating antibiotic intracellular infections
e.g. Listeria monocytogenes Ciprofloxacin resistance PhD. defense :
Maarten Aerts, May 6 th 2010Ciprofloxacin resistance
- Slide 64
- Intracellular accumulating antibiotic intracellular infections
e.g. Listeria monocytogenes Ciprofloxacin resistance PhD. defense :
Maarten Aerts, May 6 th 2010Ciprofloxacin resistance
- Slide 65
- Intracellular accumulating antibiotic Long-term exposure to
ciprofloxacin eukaryotic resistance Ciprofloxacin resistance PhD.
defense : Maarten Aerts, May 6 th 2010Ciprofloxacin resistance
- Slide 66
- Intracellular accumulating antibiotic Long-term exposure to
ciprofloxacin eukaryotic resistance Ciprofloxacin resistance PhD.
defense : Maarten Aerts, May 6 th 2010Ciprofloxacin resistance
- Slide 67
- Intracellular accumulating antibiotic Long-term exposure to
ciprofloxacin eukaryotic resistance Ciprofloxacin resistance PhD.
defense : Maarten Aerts, May 6 th 2010
- Slide 68
- Intracellular accumulating antibiotic Long-term exposure to
ciprofloxacin eukaryotic resistance Ciprofloxacin resistance PhD.
defense : Maarten Aerts, May 6 th 2010
- Slide 69
- Intracellular accumulating antibiotic Long-term exposure to
ciprofloxacin eukaryotic resistance Ciprofloxacin resistance PhD.
defense : Maarten Aerts, May 6 th 2010
- Slide 70
- Intracellular accumulating antibiotic Long-term exposure to
ciprofloxacin eukaryotic resistance Ciprofloxacin resistance PhD.
defense : Maarten Aerts, May 6 th 2010Ciprofloxacin resistance
- Slide 71
- Intracellular accumulating antibiotic Long-term exposure to
ciprofloxacin eukaryotic resistance Ciprofloxacin resistance ATP
PhD. defense : Maarten Aerts, May 6 th 2010
- Slide 72
- Intracellular accumulating antibiotic Long-term exposure to
ciprofloxacin eukaryotic resistance Ciprofloxacin resistance ATP
PhD. defense : Maarten Aerts, May 6 th 2010
- Slide 73
- Intracellular accumulating antibiotic Long-term exposure to
ciprofloxacin eukaryotic resistance Ciprofloxacin resistance MK571
PhD. defense : Maarten Aerts, May 6 th 2010
- Slide 74
- 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 Ciprofloxacin resistance
PhD. defense : Maarten Aerts, May 6 th 2010Ciprofloxacin
resistance
- Slide 75
- Comparative proteomics PhD. defense : Maarten Aerts, May 6 th
2010
- Slide 76
- Comparative proteomics WT Heavy labeled CIP Light labeled
Tryptic digest LC-MS PhD. defense : Maarten Aerts, May 6 th
2010
- Slide 77
- Comparative proteomics WT Heavy labeled CIP Light labeled
Tryptic digest LC-MS m/z PhD. defense : Maarten Aerts, May 6 th
2010
- Slide 78
- Comparative proteomics PhD. defense : Maarten Aerts, May 6 th
2010Ciprofloxacin resistance
- Slide 79
- Comparative proteomics PhD. defense : Maarten Aerts, May 6 th
2010Ciprofloxacin resistance
- Slide 80
- 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 6 th
2010Ciprofloxacin resistance
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- Discussion MRP4 Confirmed : - RT-PCR (mRNA) - Western blot
(protein) PhD. defense : Maarten Aerts, May 6 th 2010Ciprofloxacin
resistance
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- Discussion MRP4 (ABCC4) p58 IPK : Negative regulator of UPR
evoked by ER stress CIP resistance ~ Stress resistance PhD. defense
: Maarten Aerts, May 6 th 2010Ciprofloxacin resistance
- Slide 83
- Discussion MRP4 (ABCC4) p58 IPK : Negative regulator of UPR
evoked by ER stress CIP resistance ~ Stress resistance PhD. defense
: Maarten Aerts, May 6 th 2010Ciprofloxacin resistance
- Slide 84
- Discussion MRP4 (ABCC4) p58 IPK : 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 6 th 2010Ciprofloxacin resistance
- Slide 85
- Discussion Chr. 14 mrp4 p58 IPK Gene duplication PhD. defense :
Maarten Aerts, May 6 th 2010Ciprofloxacin resistance
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- Discussion MRP4 (ABCC4) p58 IPK : Negative regulator of UPR
evoked by ER stress Remaining proteins : related to drug resistance
? secondary effects ? MRP4 : cAMP, Leukotriens, etc. p58 IPK :
regulator of Transcription Factors PhD. defense : Maarten Aerts,
May 6 th 2010Ciprofloxacin resistance
- Slide 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 6 th
2010Ciprofloxacin resistance
- Slide 88
- Conclusion PhD. defense : Maarten Aerts, May 6 th
2010Ciprofloxacin resistance
- Slide 89
- Conclusion PhD. defense : Maarten Aerts, May 6 th
2010Ciprofloxacin resistance
- Slide 90
- General Conclusions PhD. defense : Maarten Aerts, May 6 th
2010Conclusions
- Slide 91
- 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 General Conclusions PhD. defense : Maarten Aerts,
May 6 th 2010Conclusions
- Slide 92
- 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 General Conclusions PhD. defense : Maarten
Aerts, May 6 th 2010Conclusions
- Slide 93
- 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 Future
perspectives PhD. defense : Maarten Aerts, May 6 th 2010Future
perspectives
- Slide 94
- Promotor : Prof. Dr. B. Devreese Co-Promotor : Prof. Dr. J. Van
Beeumen Friends and Family L-ProBE colleagues Acknowledgement
Laboratory of Zoophysiology : prof. Dr. D. de Graaf Marleen Brunain
Ellen Danneels Ellen Formesyn Unit de Pharamcologie Cellulaire et
Molculaire (UCL) : prof. Dr. P. Tulkens prof. Dr. F. Van Bambeke
Nancy Caceres Beatrice Marquez PhD. defense : Maarten Aerts, May 6
th 2010Acknowlegment
- Slide 95
- PhD. defense : Maarten Aerts, May 6 th 2010 ? Questions