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 6 th 2010

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

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

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

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

7. Central Dogma DNA mRNA Protein Proteomics : Introduction Introduction P Transcription Translation Duplication PhD. defense : Maarten Aerts, May 6 th 2010

8. Proteins : - amino acid polymer  structure and function - define a cell’s phenotype - molecular ‘active’ molecules Structural proteins Transport proteins Enzymes Proteomics : Introduction IntroductionPhD. defense : Maarten Aerts, May 6 th 2010

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

10. Proteomics : Introduction Introduction Genomics Proteomics Transcriptomics Gene function Gene prediction Gene function PhD. defense : Maarten Aerts, May 6 th 2010

11. 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

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

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

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

15. Sample Peptides Proteins Proteomics : mass spectrometry PhD. defense : Maarten Aerts, May 6 th 2010Introduction

16. Sample Peptides Proteins Ionization ESI MALDI Proteomics : mass spectrometry PhD. defense : Maarten Aerts, May 6 th 2010Introduction

17. 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

18. 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

19. Proteomics : mass spectrometry PhD. defense : Maarten Aerts, May 6 th 2010Introduction

20. 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

21. 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

22. 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

23. 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

24. 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

25. 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

26. 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

27. 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

28. 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

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

30. 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

31. 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

32. High-Resolution Mass Spectrometry IntroductionPhD. defense : Maarten Aerts, May 6 th 2010

33. 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

34. 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

35. 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

36. 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

37. 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

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

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

40. Protein mixture 2D-PAGE Digest on single spot containing one protein MS and MS/MS PMF or PFF pI / MW

41. Protein mixture 2D-PAGE Digest on single spot containing one protein MS and MS/MS PMF or PFF

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

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

44. 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

45. 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

46. 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

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

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

49. Nasonia vitripennis Ectoparasitoid wasp Nasonia Venomics : Introduction Nasonia VenomicsPhD. defense : Maarten Aerts, May 6 th 2010

50. Nasonia vitripennis Ectoparasitoid wasp Nasonia Venomics : Introduction Nasonia VenomicsPhD. defense : Maarten Aerts, May 6 th 2010

51. Nasonia vitripennis Ectoparasitoid wasp Nasonia Venomics : Introduction Nasonia VenomicsPhD. defense : Maarten Aerts, May 6 th 2010

52. Nasonia vitripennis Ectoparasitoid wasp Nasonia Venomics : Introduction Nasonia VenomicsPhD. defense : Maarten Aerts, May 6 th 2010

53. 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

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

55. 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

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

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

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

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

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

61. Case Study 2 : Ciprofloxacin resistance PhD. defense : Maarten Aerts, May 6 th 2010Ciprofloxacin resistance

62. Intracellular accumulating antibiotic  intracellular infections e.g. Listeria monocytogenes Ciprofloxacin resistance PhD. defense : Maarten Aerts, May 6 th 2010Ciprofloxacin resistance

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

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

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

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

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

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

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

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

71. Intracellular accumulating antibiotic Long-term exposure to ciprofloxacin  eukaryotic resistance Ciprofloxacin resistance ATP PhD. defense : Maarten Aerts, May 6 th 2010

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

73. Intracellular accumulating antibiotic Long-term exposure to ciprofloxacin  eukaryotic resistance Ciprofloxacin resistance MK571 PhD. defense : Maarten Aerts, May 6 th 2010

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

75. Comparative proteomics PhD. defense : Maarten Aerts, May 6 th 2010

76. Comparative proteomics WT Heavy labeled CIP Light labeled Tryptic digest LC-MS PhD. defense : Maarten Aerts, May 6 th 2010

77. Comparative proteomics WT Heavy labeled CIP Light labeled Tryptic digest LC-MS m/z PhD. defense : Maarten Aerts, May 6 th 2010

78. Comparative proteomics PhD. defense : Maarten Aerts, May 6 th 2010Ciprofloxacin resistance

79. Comparative proteomics PhD. defense : Maarten Aerts, May 6 th 2010Ciprofloxacin resistance

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

81. Discussion MRP4 Confirmed : - RT-PCR (mRNA) - Western blot (protein) PhD. defense : Maarten Aerts, May 6 th 2010Ciprofloxacin resistance

82. 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

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

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

85. Discussion Chr. 14 mrp4 p58 IPK  Gene duplication PhD. defense : Maarten Aerts, May 6 th 2010Ciprofloxacin resistance

86. 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

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

88. Conclusion PhD. defense : Maarten Aerts, May 6 th 2010Ciprofloxacin resistance

89. Conclusion PhD. defense : Maarten Aerts, May 6 th 2010Ciprofloxacin resistance

90. General Conclusions PhD. defense : Maarten Aerts, May 6 th 2010Conclusions

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

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

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

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 Unité de Pharamcologie Cellulaire et Moléculaire (UCL) : prof. Dr. P. Tulkens prof. Dr. F. Van Bambeke Nancy Caceres Beatrice Marquez PhD. defense : Maarten Aerts, May 6 th 2010Acknowlegment

95. PhD. defense : Maarten Aerts, May 6 th 2010 ? Questions