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

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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 6th 2010
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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
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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
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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
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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
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Proteomics Introduction

• Central Dogma
• DNA
• mRNA
• Protein

P
Duplication
Transcription
Translation
Introduction
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Proteomics Introduction

• Proteins
• - amino acid polymer ? structure and function
• - define a cells phenotype
• - molecular active molecules
• Structural proteins
• Transport proteins
• Enzymes

Introduction
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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
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Proteomics Introduction
Introduction
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Proteomics Introduction
micro array fast and complete
?
slow and incomplete coverage
Introduction
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Proteomics Introduction

• Central Dogma
• DNA
• mRNA
• Protein

R
P
Duplication
Transcription
R
PTM Localization
R
Translation
Introduction
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Proteomics Introduction
micro array fast and complete
slow and incomplete coverage
Introduction
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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
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Proteomics mass spectrometry
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Introduction
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Proteomics mass spectrometry
ESI MALDI
Ionization
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Introduction
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Proteomics mass spectrometry
ESI MALDI
Ionization
Quadrupole Ion Trap TOF FT-ICR Orbitrap Ion
Mobility
Mass analyzer
Detector
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Introduction
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Proteomics mass spectrometry
ESI MALDI
Ionization
Quadrupole Ion Trap TOF FT-ICR Orbitrap Ion
Mobility
Mass analyzer
Detector
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Introduction
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Proteomics mass spectrometry
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Introduction
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Proteomics Protein/Peptide Identification
Trypsin K/R
MS
Unique Peptide Mass Fingerprint
Protein database ? prediction PMF
Introduction
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Proteomics Protein/Peptide Identification
Trypsin K/L
MS
Unique Peptide Fragment Fingerprint
MS/MS
Unique Peptide Mass Fingerprint
Introduction
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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High-Resolution Mass Spectrometry
Introduction
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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
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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
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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
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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
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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
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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
• Data-Dependent Acquisition
• ? Ideal LC-MS platform
• high quality MS
• high quantity MS/MS

PhD. defense Maarten Aerts, May 6th 2010
Introduction
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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
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pI / MW
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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
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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
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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
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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
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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
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Nasonia Venomics Introduction

• Nasonia vitripennis
• Ectoparasitoid wasp

Nasonia Venomics
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Nasonia Venomics Introduction

• Nasonia vitripennis
• Ectoparasitoid wasp

Nasonia Venomics
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Nasonia Venomics Introduction

• Nasonia vitripennis
• Ectoparasitoid wasp

Nasonia Venomics
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Nasonia Venomics Introduction

• Nasonia vitripennis
• Ectoparasitoid wasp

Nasonia Venomics
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Nasonia Venomics Introduction

• Nasonia vitripennis
• Ectoparasitoid wasp

Venom proteins - Host behavior - Growth
alteration - Immune suppression - Nutrient
metabolism
Nasonia Venomics
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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
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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
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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
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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
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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
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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
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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
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Case Study 2 Ciprofloxacin resistance
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Ciprofloxacin resistance
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Ciprofloxacin resistance

• Intracellular accumulating antibiotic
• ? intracellular infections e.g. Listeria
  monocytogenes

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Ciprofloxacin resistance
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Ciprofloxacin resistance

• Intracellular accumulating antibiotic
• ? intracellular infections e.g. Listeria
  monocytogenes

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Ciprofloxacin resistance
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Ciprofloxacin resistance

• Intracellular accumulating antibiotic
• ? intracellular infections e.g. Listeria
  monocytogenes

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Ciprofloxacin resistance
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Ciprofloxacin resistance

• Intracellular accumulating antibiotic
• Long-term exposure to ciprofloxacin ? eukaryotic
  resistance

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Ciprofloxacin resistance
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Ciprofloxacin resistance

• Intracellular accumulating antibiotic
• Long-term exposure to ciprofloxacin ? eukaryotic
  resistance

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Ciprofloxacin resistance
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Ciprofloxacin resistance

• Intracellular accumulating antibiotic
• Long-term exposure to ciprofloxacin ? eukaryotic
  resistance

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Ciprofloxacin resistance

• Intracellular accumulating antibiotic
• Long-term exposure to ciprofloxacin ? eukaryotic
  resistance

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Ciprofloxacin resistance

• Intracellular accumulating antibiotic
• Long-term exposure to ciprofloxacin ? eukaryotic
  resistance

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Ciprofloxacin resistance

• Intracellular accumulating antibiotic
• Long-term exposure to ciprofloxacin ? eukaryotic
  resistance

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Ciprofloxacin resistance
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Ciprofloxacin resistance

• Intracellular accumulating antibiotic
• Long-term exposure to ciprofloxacin ? eukaryotic
  resistance

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Ciprofloxacin resistance

• Intracellular accumulating antibiotic
• Long-term exposure to ciprofloxacin ? eukaryotic
  resistance

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Ciprofloxacin resistance

• Intracellular accumulating antibiotic
• Long-term exposure to ciprofloxacin ? eukaryotic
  resistance

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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

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Ciprofloxacin resistance
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Comparative proteomics
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Comparative proteomics
WT Heavy labeled
CIP Light labeled
Tryptic digest
LC-MS
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Comparative proteomics
WT Heavy labeled
CIP Light labeled
Tryptic digest
LC-MS
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Comparative proteomics
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Ciprofloxacin resistance
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Comparative proteomics
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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

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Ciprofloxacin resistance
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Discussion
MRP4
Confirmed - RT-PCR (mRNA) - Western blot
(protein)
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Ciprofloxacin resistance
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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
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Discussion

• MRP4 (ABCC4)
• p58IPK Negative regulator of UPR evoked by ER
  stress
• ? CIP resistance Stress resistance

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Ciprofloxacin resistance
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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
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Discussion
Chr. 14
mrp4
p58IPK
? Gene duplication
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Ciprofloxacin resistance
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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

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Ciprofloxacin resistance
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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

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Ciprofloxacin resistance
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Conclusion
PhD. defense Maarten Aerts, May 6th 2010
Ciprofloxacin resistance
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Conclusion
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Ciprofloxacin resistance
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General Conclusions
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Conclusions
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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

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Conclusions
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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

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Conclusions
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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

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Future perspectives
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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
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Acknowlegment
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?
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Questions