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Study of Lyme disease using proteomic approach

Study of Lyme disease using proteomic approach. Westermann Benoît Student Symposium UMR 7178 - DSA - Laboratoire de Spectrométrie de Masse BioOrganique. Introduction. Host. What is Lyme disease? Vector-borne transmission disease which is the most widespread in North hemisphere.

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Study of Lyme disease using proteomic approach

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  1. Study of Lyme disease using proteomic approach Westermann Benoît Student Symposium UMR 7178 - DSA - Laboratoire de Spectrométrie de Masse BioOrganique

  2. Introduction Host • What is Lyme disease? • Vector-borne transmission disease which is the most widespread in North hemisphere 65 000 new cases/year in Europe 3 000 new cases/year in Alsace Pathogen Bacteria Borrelia burgdorferi sensu lato Vector Tick Ixodes Triad of interactions Student Symposium _ Westermann Benoît _ 31 octobre 2013

  3. Introduction Host • What is Lyme disease? • Vector-borne transmission disease which is the most widespread in North hemisphere 65 000 new cases/year in Europe 3 000 new cases/year in Alsace Pathogen Bacteria Borrelia burgdorferi sensu lato Vector Tick Ixodes Triad of interactions Neuroborreliosis Migrant erithema Arthritis Student Symposium _ Westermann Benoît _ 31 octobre 2013

  4. Introduction Host • What is Lyme disease? • Vector-borne transmission disease which is the most widespread in North hemisphere 65 000 new cases/year in Europe 3 000 new cases/year in Alsace Pathogen Bacteria Borrelia burgdorferi sensu lato Vector Tick Ixodes Triad of interactions  No human vaccine / Some animal vaccines  Lack of early diagnosis Search for proteins implied in the virulence of the bacteria and in the transmission of the disease Collaboration with Institut de Bactériologie Student Symposium _ Westermann Benoît _ 31 octobre 2013

  5. Preliminary studies : in vitro analyses of Borrelia Genus Pathogen species Strain Strong virulence B. garinii Weak virulence Strong virulence B. afzelii B. burgdorferisl Weak virulence Strong virulence B. burgdorferiss Weak virulence Student Symposium _ Westermann Benoît _ 31 octobre 2013

  6. Preliminary studies : in vitro analyses of Borrelia Genus Pathogen species Strain Strong virulence B. garinii Weak virulence Strong virulence B. afzelii B. burgdorferisl Weak virulence Strong virulence B. burgdorferiss Weak virulence Exemple for B. burgdorferi ss In vitro culture of Borrelia v Identification of Borrelia proteins 34 790 56 Specific or overexpressed proteins identified in the strong virulence strain (done by Gilles Schnell) Weak virulence (824 proteins) Strong virulence (846 proteins) Student Symposium _ Westermann Benoît _ 31 octobre 2013

  7. Preliminary studies : in vitro analyses of Borrelia Genus Pathogen species Strain Strong virulence B. garinii Weak virulence Strong virulence B. afzelii B. burgdorferisl Weak virulence Strong virulence B. burgdorferiss Weak virulence Exemple for B. burgdorferi ss In vitro culture of Borrelia v Identification of Borrelia proteins 34 790 56 Specific or overexpressed proteins identified in the strong virulence strain Selection of 40 specific or overexpressed proteins Weak virulence (824 proteins) Strong virulence (846 proteins) Student Symposium _ Westermann Benoît _ 31 octobre 2013

  8. Preliminary studies : in vitro analyses of Borrelia Differences between invitro / invivo ? 1. Identification of Borrelia proteins expressed in vivowith the three pathogen species Inter-species comparison and selection of the target proteins Vaccine 2. Diagnostic Development of a new targeted detection method of Borrelia proteins (using LC-SRM) Student Symposium _ Westermann Benoît _ 31 octobre 2013

  9. 1. In vivo identification of proteins _ Analytical strategy Borrelia inoculation for each species in mouse (Strong / Weak strain) Sampling skin biopsies at D+5 and D+7 (peak multiplication of bacteria in skin) Student Symposium _ Westermann Benoît _ 31 octobre 2013

  10. 1. In vivo identification of proteins _ Analytical strategy Proteins extraction of infected mouse skin biopsies (4 mg of skin) Inoculation of Borrelia Incubation Sampling skin biopsies Student Symposium _ Westermann Benoît _ 31 octobre 2013

  11. 1. In vivo identification of proteins _ Analytical strategy Molecular Markers Track C D+7 (2) Track B D+7 (1) Track A D+5 250 150 100 75 50 37 25 20 KDa SDS-PAGE 12% Direction of migration Inoculation of Borrelia Incubation Sampling skin biopsies Proteins extraction Loading proteins on a electrophoresis gel (SDS-PAGE) Proteins migration according to their molecular weight Systematic excision of 25 bands Student Symposium _ Westermann Benoît _ 31 octobre 2013

  12. 1. In vivo identification of proteins _ Analytical strategy Track C D+7 (2) Track B D+7 (1) Track A D+5 Molecular Markers 250 150 100 75 50 37 25 20 KDa SDS-PAGE 12% Direction of migration Inoculation of Borrelia Incubation Sampling skin biopsies Proteins extraction Gel separation (SDS-PAGE at 12%) Bands excision Reduction (DTT) Alkylation (IAA) Tryptic ingel digestion Extraction of peptides -S-S- -S-H R R -S-Cam K K Student Symposium _ Westermann Benoît _ 31 octobre 2013

  13. 1. In vivo identification of proteins _ Analytical strategy Track C D+7 (2) Track B D+7 (1) Track A D+5 Molecular Markers 250 150 100 75 50 37 25 20 KDa SDS-PAGE 12% Direction of migration Inoculation of Borrelia Incubation Sampling skin biopsies Proteins extraction Gel separation (SDS-PAGE at 12%) Bands excision Intens. x109 Nano chromatography coupled to a mass spectrometer (MS /MS) Base PeakChromatogram -S-S- 3 2 -S-H 1 R Zorbax 300SB-C18 (150 mm, 75 mm, flow rate 300 nL/min) Gradient : acetonitrile with 0,1% FA R -S-Cam K K 0 Tryptic ingel digestion 0 5 10 15 20 25 30 Time [min] Student Symposium _ Westermann Benoît _ 31 octobre 2013

  14. 1. In vivo identification of proteins _ Analytical strategy Track C D+7 (2) Track B D+7 (1) Track A D+5 Molecular Markers 250 150 100 75 50 37 25 20 KDa SDS-PAGE 12% Direction of migration Inoculation of Borrelia Incubation Sampling skin biopsies Proteins extraction Gel separation (SDS-PAGE at 12%) Bands excision Intens. Data interpretation MS spectra (peptides) MS/MS spectra (fragments from the peptides) x109 Base PeakChromatogram -S-S- 3 2 -S-H 1 R R -S-Cam K K 0 Tryptic ingel digestion 0 5 10 15 20 25 30 Time [min] Nano chromatography coupled to a mass spectrometer Student Symposium _ Westermann Benoît _ 31 octobre 2013

  15. 1. In vivo identification of proteins _ Analytical strategy Track C D+7 (2) Track B D+7 (1) Track A D+5 Molecular Markers 250 150 100 75 50 37 25 20 KDa SDS-PAGE 12% Direction of migration Inoculation of Borrelia Incubation Sampling skin biopsies Proteins extraction Gel separation (SDS-PAGE at 12%) Bands excision Intens. x109 Base PeakChromatogram -S-S- 3 Proteins identification 2 -S-H 1 R R -S-Cam K K Data interpretation 0 Tryptic ingel digestion 0 5 10 15 20 25 30 Time [min] Nano chromatography coupled to a mass spectrometer Student Symposium _ Westermann Benoît _ 31 octobre 2013

  16. 1. In vivo identification of proteins _ Results • Approximately 1350 mouse proteins identified for each infected mouse skin biopsies • Borrelia proteins represent less than 1% of all identified proteins • Proteins implied in the virulence of the bacteria : OspC, DbpA, BmpA • Others : Flagelline, Hypothetical proteins, … Student Symposium _ Westermann Benoît _ 31 octobre 2013

  17. 1. In vivo identification of proteins _ Results • Approximately 1350 mouse proteins identified for each infected mouse skin biopsies • Borrelia proteins represent less than 1% of all identified proteins • Proteins implied in the virulence of the bacteria : OspC, DbpA, BmpA • Others : Flagelline, Hypothetical proteins, … Interesting proteins for diagnosis of Lyme disease (using targeted mass spectrometry, LC-SRM) Student Symposium _ Westermann Benoît _ 31 octobre 2013

  18. 2. Targeted detection of in vivo proteins _ Analytical strategy LC-SRM = Liquid Chromatography-Selected Reaction Monitoring Intensity Q3 LC Q1 Q2 Q3 Time Intensity Proteins Selection of precusor ion Fragmentation induced by collision Selection of fragment ion(s) m/z Selection of transitions  Specific and sensible technique Precursor ion = prototypic peptide of Borrelia Student Symposium _ Westermann Benoît _ 31 octobre 2013

  19. 2. Targeted detection of in vivo proteins _ Analytical strategy LC-SRM = Liquid Chromatography-Selected Reaction Monitoring Intensity Q3 LC Q1 Q2 Time Intensity Proteins Selection of precusor ion Fragmentation induced by collision Selection of fragment ion(s) m/z Selection of transitions  Specific and sensible technique Precursor ion = prototypic peptide Synthetic peptide isotopically marked (+8 Da or + 10 Da)  same physico-chemical properties  Validation and quantification of endogene peptides Student Symposium _ Westermann Benoît _ 31 octobre 2013

  20. 2. Targeted detection of in vivo proteins _ Results • Analysis of infected mouse skin biopsy by B. burgdorferi ss Reduction (DTT) Alkylation (IAA) Trypsic digestion Peptides extraction B. burgdorferiss Inoculation Sampling skin biopsies Proteins extraction SDS-PAGE Bands excision LC-SRM 250 150 100 75 50 37  25 20 KDa R R K K Spike with heavy peptides Student Symposium _ Westermann Benoît _ 31 octobre 2013

  21. 2. Targeted detection of in vivo proteins _ Results • Analysis of infected mouse skin biopsy by B. burgdorferi ss Reduction (DTT) Alkylation (IAA) Trypsic digestion Peptides extraction B. burgdorferiss Inoculation Sampling skin biopsies Proteins extraction SDS-PAGE Bands excision LC-SRM 250 150 100 75 50 37  25 20 KDa R R K K Spike with heavy peptides Detection of 4 Borrelia proteins (Flagelline, OspC, DbpA, GADPH) Quantification of OspC 302 fmol/mg of biopsie Student Symposium _ Westermann Benoît _ 31 octobre 2013

  22. 3. Biological reproducibility study • Principle of immunodetection by Western Blot Molecular Markers C B A B. burgdorferiss Inoculation Sampling skin biopsies Proteins extraction SDS-PAGE Bands excision 250 150 100 75 50 37 25 20 KDa SDS-PAGE 12% Transfer of proteins on nitrocellulose membrane under electric field Student Symposium _ Westermann Benoît _ 31 octobre 2013

  23. 3. Biological reproducibility study • Principle of immunodetection by Western Blot Molecular Markers C B A B. burgdorferiss Inoculation Sampling skin biopsies Proteins extraction SDS-PAGE Bands excision 250 150 100 75 50 37 25 20 KDa Transfert of proteins on nitrocellulose membrane SDS-PAGE 12% Loading Ab I directed against targeted protein Ab I Targetedprotein Membrane Student Symposium _ Westermann Benoît _ 31 octobre 2013

  24. 3. Biological reproducibility study • Principle of immunodetection by Western Blot Molecular Markers C B A B. burgdorferiss Inoculation Sampling skin biopsies Proteins extraction SDS-PAGE Bands excision 250 150 100 75 50 37 25 20 KDa Transfert of proteins on nitrocellulose membrane SDS-PAGE 12% Loading Ab II anti-IgG  Contain a reactive enzyme : HRP Ab I Targeted protein Membrane Ab I directed against targeted protein Student Symposium _ Westermann Benoît _ 31 octobre 2013

  25. 3. Biological reproducibility study • Principle of immunodetection by Western Blot Molecular Markers C B A B. burgdorferiss Inoculation Sampling skin biopsies Proteins extraction SDS-PAGE Bands excision 250 150 100 75 50 37 25 20 KDa Transfert of proteins on nitrocellulose membrane SDS-PAGE 12% Ab I Targeted protein Membrane Ab II anti-IgG Ab I against targeted protein Revelation on radiographic film Peak intensities integration Student Symposium _ Westermann Benoît _ 31 octobre 2013

  26. 3. Biological reproducibility study B B B A A A • 3 infected mouses and for each mouse 3 skin biopsies : C C C Actin (Mouse) Mouse 1 Mouse 2 Mouse 3 OspC (Borrelia) Student Symposium _ Westermann Benoît _ 31 octobre 2013

  27. 3. Biological reproducibility study B B B A A A • 3 infected mouses and for each mouse 3 skin biopsies : C C C Actin (Mouse) Mouse 1 Mouse 2 Mouse 3 OspC (Borrelia) • Low inter-mouse variation of actin intensities •  Good experimental reproducibility (protein quantification, SDS-PAGE gel, transfer, revelation) • The intra-mouse OspC variability is between 30% and 60% •  These results were confirmed by targeted mass spectrometry (LC-SRM) and by biologists (RT-PCR) Student Symposium _ Westermann Benoît _ 31 octobre 2013

  28. 4. Targeted detection of in vivo proteins _ Resultat • Analysis of human infected skin biopsie (after tick bite) Reduction (DTT) Alkylation (IAA) Trypsic digestion Peptides extraction Borrelia infection Sampling human skin biopsy Proteins extraction SDS-PAGE Bands excision LC-SRM 250 150 100 75 50 37 25 20 KDa R R K K Spike with heavy peptides Detection of OspC in the 3 analysed infected human skin biopsies Student Symposium _ Westermann Benoît _ 31 octobre 2013

  29. Outlooks • Analyze more infected human skin biopsies • Develop a new sample preparation method without the use of electrophoresis gel Student Symposium _ Westermann Benoît _ 31 octobre 2013

  30. Identification des protéines par méthode PFF Spectres MS/MS Banques protéiques CCFDSDFRGTHTTYIPLM CCFDSDFRGTHTTYIPLM DFFSDFFIRLFPFMLDKF DFFSDFFIRLFPFMLDKF DFHEGRILPMKYITRFNC DFHEGRILPMKYITRFNC RKMDEASCVNFJRPQSD RKMDEASCVNFJRPQSD FDSFIRLFPFMLDKFNVC FDSFIRLFPFMLDKFNVC NVCGDFSREFDGEDGGD NVCGDFSREFDGEDGGD VFGNDFHEGRILPMKYIT VFGNDFHEGRILPMKYIT HDSFAQRSFEDGEHDIRL HDSFAQRSFEDGEHDIRL GHLKITYPMNVCDSDFR GHLKITYPMNVCDSDFR IRLFPFMLDKFNVCGDFS IRLFPFMLDKFNVCGDFS GDFSREFDGEDGGDFGF GDFSREFDGEDGGDFGF FGFHEHSDGDTSGQFAR FGFHEHSDGDTSGQFAR RFEDDFHEGRILPMKYIT RFEDDFHEGRILPMKYIT RFNCHDSFAQRSFEDGE RFNCHDSFAQRSFEDGE FPMTGFKNVDFCGERSF FPMTGFKNVDFCGERSF Digestion trypsique et fragmentation in silico AQSDFERTGGHPLMNVF AQSDFERTGGHPLMNVF REFDGEDGGDFGFHEHS REFDGEDGGDFGFHEHS HEHSDGDTSGQFARSFE HEHSDGDTSGQFARSFE SFERDFCVVNFKFLDIKD SFERDFCVVNFKFLDIKD IRMLKLFPFMLDKFNVC IRMLKLFPFMLDKFNVC FGEGRILPMKYITRFNCH FGEGRILPMKYITRFNCH RFNCHDSFAQRSFEDGE RFNCHDSFAQRSFEDGE HDIRLFPMTGFKNVDFC HDIRLFPMTGFKNVDFC DGCVAQDHFIRLPFMFY DGCVAQDHFIRLPFMFY FRDSQACVSDEFRHKITP FRDSQACVSDEFRHKITP DGDTSGQFARSFERDFC DGDTSGQFARSFERDFC RDFCVVNFKFLDIKDLFL RDFCVVNFKFLDIKDLFL LFLFMPRLGFHHTFLFIK LFLFMPRLGFHHTFLFIK GDFSREFDGEDGGDFGF GDFSREFDGEDGGDFGF DSFAQRSFEDGEHDIRLF DSFAQRSFEDGEHDIRLF HDIRLFPMTGFKNVDFC HDIRLFPMTGFKNVDFC GERSFDGCVAQDHFIRLP GERSFDGCVAQDHFIRLP RHGTTDGNVHCFSQDAE RHGTTDGNVHCFSQDAE LFMDLMFPRTEQASSCV LFMDLMFPRTEQASSCV VVNFKFLDIKDLFLFMPR VVNFKFLDIKDLFLFMPR FMPRLGFHHTFLFIKDKG FMPRLGFHHTFLFIKDKG DKGHRDGFTEGDHSGDF DKGHRDGFTEGDHSGDF HEHSDGDTSGQFARSFE HEHSDGDTSGQFARSFE PMTGFKNVDFCGERSFD PMTGFKNVDFCGERSFD GERSFDGCVAQDHFIRLP GERSFDGCVAQDHFIRLP FMFYRHGTTDGNVHCFS FMFYRHGTTDGNVHCFS QDSFEGDTHFFKGFLFM QDSFEGDTHFFKGFLFM FHDNRTIPLYMTIFKGSR FHDNRTIPLYMTIFKGSR LGFHHTFLFIKDKGHRD LGFHHTFLFIKDKGHRD HRDGFTEGDHSGDFHCD HRDGFTEGDHSGDFHCD HCDPMDLCNCDSREFDD HCDPMDLCNCDSREFDD RDFCVVNFKFLDIKDLFL RDFCVVNFKFLDIKDLFL GCVAQDHFIRLPFMFYR GCVAQDHFIRLPFMFYR FMFYRHGTTDGNVHCFS FMFYRHGTTDGNVHCFS QDAEQDSFEGDTHFFKG QDAEQDSFEGDTHFFKG GPITLFGKITRYFHGNVD GPITLFGKITRYFHGNVD EFDYAGSRHEYD EFDYAGSRHEYD GFTEGDHSGDFHCDPMD GFTEGDHSGDFHCDPMD PMDLCNCDSREFDDSGG PMDLCNCDSREFDDSGG SGGGQFASRAQFSRSDQ SGGGQFASRAQFSRSDQ FMPRLGFHHTFLFIKDKG FMPRLGFHHTFLFIKDKG HGTTDGNVHCFSQDAEQ HGTTDGNVHCFSQDAEQ QDAEQDSFEGDTHFFKG QDAEQDSFEGDTHFFKG FLFMGPITLFGKITRYFH FLFMGPITLFGKITRYFH FSQREAS FSQREAS LCNCDSREFDDSGGGQF LCNCDSREFDDSGGGQF GQFASRAQFSRSDQDIPM GQFASRAQFSRSDQDIPM DIPMDLIRKFMPLCCFD DIPMDLIRKFMPLCCFD HRDGFTEGDHSGDFHCD HRDGFTEGDHSGDFHCD DSFEGDTHFFKGFLFMG DSFEGDTHFFKGFLFMG FLFMGPITLFGKITRYFH FLFMGPITLFGKITRYFH GNVDFSQREAS GNVDFSQREAS ASRAQFSRSDQDIPMDLI ASRAQFSRSDQDIPMDLI DLIRKFMPLCCFD DLIRKFMPLCCFD PMDLCNCDSREFDDSGG PMDLCNCDSREFDDSGG PITLFGKITRYFHGNVDF PITLFGKITRYFHGNVDF GNVDFSQREAS GNVDFSQREAS RKFMPLCCFD RKFMPLCCFD GQFASRAQFSRSDQDIPM GQFASRAQFSRSDQDIPM SQREAS SQREAS DLIRKFMPLCCFD DLIRKFMPLCCFD Listes de masses expérimentales Listes de masses théoriques MS et MS/MS Protéine à identifier Protéine A Protéine B Protéine C MS MS/MS MS MS/MS MS MS/MS MS MS/MS 789,67 756,24 789,67 876,43 756,24 854,25 Comparaison des listes de masses 854,25 987,94 876,43 956,36 956,36 987,49 999,22 998,65 998,65 999,12 1002,13 1018,98 546,45 1002,13 589,36 1103,26 1342,34 1018,98 569,36 546,45 1103,26 1230,65 1342,34 1597,09 1230,65 1523,58 1597,09 1678,97 1523,58 1658,75 1987,65 1678,97 1856,26 1856,26 1987,65 1956,24 1956,24 2202,22 2018,80 516,90 895,36 895,36 516,90 634,79 958,29 958,29 612,75 634,79 Mascot / OMSSA 999,36 999,36 612,75 752,93 1002,35 1002,35 752,93 1125,25 879,89 955,69 1125,25 785,69 678,09 1254,36 999,78 879,89 1254,36 1633,25 678,09 999,78 1633,25 1134,48 1785,36 1785,36 1134,48 1368,84 1985,59 1985,59 1596,28 1368,84 2015,69 2015,69 1596,28 1675,89 1888,44 1675,89 1888,44 759,26 759,26 895,32 712,43 895,32 985,36 712,43 750,09 985,36 999,25 812,34 750,09 999,25 1561,56 1012,26 812,34 1012,26 933,66 1001,56 1203,25 1203,25 933,66 978,55 1259,36 1259,36 1207,45 978,55 1526,35 1002,56 1526,35 1207,45 1327,87 1965,25 1002,56 1965,25 2015,26 1327,87 1387,23 2015,26 Identification protéine Puis validation 1505,72 1387,23 1505,72 1678,06 1835,79 1678,06 1835,79 1944,55 1944,55 Student Symposium _ Westermann Benoît _ 31 octobre 2013

  31. Exemple de spectre MS/MS Student Symposium _ Westermann Benoît _ 31 octobre 2013

  32. Critères d’acceptation du signal en LC-SRM • La détection en SRM se base sur plusieurs paramètres •  co-élution des transitions suivies •  co-élution des peptides lourd et léger •  respect du ratio des transitions Co-élution des transitions suivies Co-élution entre le peptide lourd (bleu) et endogène (rouge) Respect du ratio des transitions Student Symposium _ Westermann Benoît _ 31 octobre 2013

  33. Cycle d’infection par la bactérie B. burgdorferisl Radolf J. et al, Nature ReviewsMicrobiology, 2012 Student Symposium _ Westermann Benoît _ 31 octobre 2013

  34. Schéma d’un piège à ions Gaz tampon : Argon Student Symposium _ Westermann Benoît _ 31 octobre 2013

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