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Capturing the essence of life<br />HX-IA Instrument™<br />ThorleifLavold<br />Biomotif AB<br />
The HX-IA Instrument™<br />H/D Exchange to studyconformation, bindingsites, EpitopeMapping etc.<br />ElectroCapture techno...
New HX-IA Instrument<br />
D2O<br />H’s & D’s<br />at backbone amide positions<br />HDX Amide Hydrogen Exchange<br />
−D<br />−D<br />−D<br />−D<br />−D<br />−D<br />−D<br />−D<br />−D<br />−D<br />−D<br />−D<br />−D<br />−D<br />−D<br />−D...
HDX Amide Hydrogen Exchange<br /><ul><li>To study dynamics of:
Protein folding &
Conformational changes
Localisation of:
Epitope
Mutation
Aggregation
Ligand binding site
Complementary information to X-ray and NMR</li></ul>vs<br />Folding Dynamics<br />?<br />Conformational Changes<br />Epito...
Membrane –AssistedSample Preparation in the HX-IA Instrument<br />Release/loop loading<br />TrypsincColumnwash.<br />LC-MS...
Membrane-Assisted Sample Preparation<br />Semipermeable membrane separates sample channel from the deuterating solution<br...
Membrane-Assisted Sample Preparation<br />Semipermeable membrane separates the sample channel from the deuterating solutio...
Amyloid β Protein Monomer HDX  <br />
Different Deuteration LevelsAccording to Protein Conformation<br />15 % Deuteration<br />PROTECTED<br />60 % Deuteration<b...
Different Deuteration LevelsAccording to Protein Conformation<br />
INTERLEIKIN 1β  /  ANTIBOBY H/D EXCHANGE EXPERIMENTS <br />
Where does it bind? Epitope mapping by MS using 1H2H exchange (HDX) or covalent modification<br />Unmodified Epitope<br />...
Conclusions<br />Online Membrane-Assisted HDX<br />No D2O Dilution              No Pippetting<br /><ul><li>  Less sample c...
  Flow Injection Deuteration (no need for automatedpipetting stations)
  No need of liquid Nitrogen (online )
Acidification on-line with no dilution of sample
  Tris or Phosphatebuffercan be used
Can be coupled with any  API-MS system </li></li></ul><li>INTERACTOMICS<br />Does it bind?How strongly does it bind?Where ...
Electrocapture-based Separations<br />Positive<br />Electrode<br />Negative<br />Electrode<br />Charge particle <br />Elec...
New Flat membrane cell <br />
New cell in PEEK<br />
AnalyticalApplications<br />Capillaryelectrophoresis<br />Protein band<br />Flow<br />MALDI & ESI-MS <br />Micro reactions...
Conductive<br />membrane<br />125 m<br />Flow<br />Peek tubing<br />Captured    protein<br />Image downstream cathode jun...
ESI Total Ion Count of an Electrocapture-concentration Experiment<br />80-fold magnification!<br />.<br />
Desalting of BSA trypticpeptides<br />MALDI-MS<br />Astorga-Wells, J., Jörnvall, H. and Bergman, T. Anal. Chem.,2003, 75,5...
Detergent Cleanup MALDI-MS<br />BSA trypticpeptides<br />Astorga-Wells, J., Jörnvall, H. and Bergman, T. Anal. Chem.,2003,...
ESI-MS Analysis of α-Lactalbumin in a solution containing 0.5% Non-Ionic Detergent   <br />Detergent clusters<br />n-octyl...
ESI-MS Analysis of α-Lactalbumin in 0.5% Non-Ionic Detergent after Electrocapture <br />Protein peaks<br />After Electroca...
Vf>Ve = ue x E<br />Vf=Ve = ue x E<br />Flow<br />200<br />E (V/cm)<br />Time<br />Capturing the essence of life<br />
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Hx Show 2011 08 31

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A new multifunctional HX-IA Instruemnt for mass spectrometry. Study H/D Exchange, structure, conformation and molecular interactions in the liquid phase.

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Transcript of "Hx Show 2011 08 31"

  1. 1. Capturing the essence of life<br />HX-IA Instrument™<br />ThorleifLavold<br />Biomotif AB<br />
  2. 2. The HX-IA Instrument™<br />H/D Exchange to studyconformation, bindingsites, EpitopeMapping etc.<br />ElectroCapture technology<br />”Findtheneedleinthehaystack” for on-line pI separation of proteins <br />Ligand Screening<br />CompoundFishing-EpitopeImprinting<br />
  3. 3.
  4. 4. New HX-IA Instrument<br />
  5. 5. D2O<br />H’s & D’s<br />at backbone amide positions<br />HDX Amide Hydrogen Exchange<br />
  6. 6. −D<br />−D<br />−D<br />−D<br />−D<br />−D<br />−D<br />−D<br />−D<br />−D<br />−D<br />−D<br />−D<br />−D<br />−D<br />−D<br />−D<br />−D<br />−D<br />−D<br />−D<br />−D<br />−D<br />−D<br />−D<br />−D<br />−D<br />−D<br />−D<br />−D<br />−D<br />−D<br />H/D Exchange for Monitoring Protein Structural Changes<br />IN HEAVY WATER<br />Intensity<br />FOLDED<br />m/z<br />mass shift<br />After 10 min<br />more D incorporated<br />IN HEAVY WATER<br />Intensity<br />UNFOLDED<br />m/z<br />
  7. 7. HDX Amide Hydrogen Exchange<br /><ul><li>To study dynamics of:
  8. 8. Protein folding &
  9. 9. Conformational changes
  10. 10. Localisation of:
  11. 11. Epitope
  12. 12. Mutation
  13. 13. Aggregation
  14. 14. Ligand binding site
  15. 15. Complementary information to X-ray and NMR</li></ul>vs<br />Folding Dynamics<br />?<br />Conformational Changes<br />Epitope Mapping<br />Mutation<br />Aggregation<br />?<br />Ligand Binding<br />?<br />?<br />Relation to X-Ray, NMR<br />
  16. 16.
  17. 17. Membrane –AssistedSample Preparation in the HX-IA Instrument<br />Release/loop loading<br />TrypsincColumnwash.<br />LC-MS analysis<br />Electrolytefluidiclines<br />Buffer and waste bottles<br />50 mM NH4CH3COO<br />100% D2O<br />10 mM NH4CH3COO<br />Gradient Pumps <br />D2O<br />D2O<br />D2O<br />Deuteration<br />Isocratic Pump <br />Autosampler<br />Srynge Pumps Power supply<br />25 mM NH4CH3COO<br />1 % Formic Ac.<br />Isocratic Pump <br />Pepsin column<br />Columnwashing<br />Valve A<br />Valve B<br />SampleElution<br />Gradient 5-95% ACN in 45min 0.1%FA<br />Pre-column<br />Loop Loading<br />Mass Spectrometer<br />Analytical Column<br />Solvent lines<br />0.05 % TFA<br />Waste lines<br />
  18. 18. Membrane-Assisted Sample Preparation<br />Semipermeable membrane separates sample channel from the deuterating solution<br />Sample<br />Channel<br />Flow<br />Semipermeable Membrane<br />Second Solution<br />Flow<br />Semi-permeable Membranes<br />
  19. 19. Membrane-Assisted Sample Preparation<br />Semipermeable membrane separates the sample channel from the deuterating solution<br />Sample<br />Channel<br />Flow<br />Membrane<br />Second Solution<br />
  20. 20. Amyloid β Protein Monomer HDX <br />
  21. 21. Different Deuteration LevelsAccording to Protein Conformation<br />15 % Deuteration<br />PROTECTED<br />60 % Deuteration<br />Unprotected<br />0 % Deuteration<br />PROTECTED<br />(Internal Pocket)<br />35 % Deuteration<br />Semi-protected<br />
  22. 22. Different Deuteration LevelsAccording to Protein Conformation<br />
  23. 23. INTERLEIKIN 1β / ANTIBOBY H/D EXCHANGE EXPERIMENTS <br />
  24. 24. Where does it bind? Epitope mapping by MS using 1H2H exchange (HDX) or covalent modification<br />Unmodified Epitope<br />Denature<br />Digestion<br />Surface Modification<br />Protein<br />Intact Mass MS<br />Ligand<br />Peptide Analysis<br />DM<br />Literature example of irreversible oxidative modification of Myoglobin<br />
  25. 25. Conclusions<br />Online Membrane-Assisted HDX<br />No D2O Dilution No Pippetting<br /><ul><li> Less sample consumption
  26. 26. Flow Injection Deuteration (no need for automatedpipetting stations)
  27. 27. No need of liquid Nitrogen (online )
  28. 28. Acidification on-line with no dilution of sample
  29. 29. Tris or Phosphatebuffercan be used
  30. 30. Can be coupled with any API-MS system </li></li></ul><li>INTERACTOMICS<br />Does it bind?How strongly does it bind?Where does it bind?<br />What happens on binding?<br />To what does it bind?<br />What binds?<br />Detection of non-covalently linked protein-ligand complexes<br />Kd determination is possible to rank ligands<br />Epitope mapping gives positional information<br />Protein consumption is low<br />Gas Phase Ion mobility shows great potential for investigating PPIs.<br />BUT…. How representative is the gas phase structure to that in solution<br />
  31. 31.
  32. 32. Electrocapture-based Separations<br />Positive<br />Electrode<br />Negative<br />Electrode<br />Charge particle <br />Electrocapture conditions will be fulfilled when<br /> Ve≥ Vf<br />Ve = Electrophoretic velocity<br />ue = Electrophoresis mobility<br />E = Electric field<br />Electrophoretic velocity is given by,<br /> Ve= ue x E<br />Capturing the essence of life<br />
  33. 33.
  34. 34. New Flat membrane cell <br />
  35. 35. New cell in PEEK<br />
  36. 36. AnalyticalApplications<br />Capillaryelectrophoresis<br />Protein band<br />Flow<br />MALDI & ESI-MS <br />Micro reactions<br />Anodic chamber<br />Cathodic chamber<br />Separations, μ-LC<br />
  37. 37. Conductive<br />membrane<br />125 m<br />Flow<br />Peek tubing<br />Captured protein<br />Image downstream cathode junction during capture-device operation<br />Protein captured at 30 nL spot<br />
  38. 38. ESI Total Ion Count of an Electrocapture-concentration Experiment<br />80-fold magnification!<br />.<br />
  39. 39. Desalting of BSA trypticpeptides<br />MALDI-MS<br />Astorga-Wells, J., Jörnvall, H. and Bergman, T. Anal. Chem.,2003, 75,5213-5219<br />
  40. 40. Detergent Cleanup MALDI-MS<br />BSA trypticpeptides<br />Astorga-Wells, J., Jörnvall, H. and Bergman, T. Anal. Chem.,2003, 75,5213-5219<br />
  41. 41. ESI-MS Analysis of α-Lactalbumin in a solution containing 0.5% Non-Ionic Detergent <br />Detergent clusters<br />n-octyl β-D glucopyranoside<br />
  42. 42. ESI-MS Analysis of α-Lactalbumin in 0.5% Non-Ionic Detergent after Electrocapture <br />Protein peaks<br />After Electrocapture<br />
  43. 43. Vf>Ve = ue x E<br />Vf=Ve = ue x E<br />Flow<br />200<br />E (V/cm)<br />Time<br />Capturing the essence of life<br />
  44. 44. Electrocapture-based Separations of Proteins <br />Beta-lactoglobulin<br />60 V<br />Beta-casein<br />90 V<br />Ribonuclease<br />120 V<br />
  45. 45. FindtheneedleinthehaystackInstrument™<br />
  46. 46. 2D-EC “NeedleInTheHaystack”<br />Green, grey and purple fractions <br />above 171 V/cm goes to waste<br />+<br />+<br />-<br />-<br />Cell #1 is held at 170V/cm Cell #2 at 171V/cm<br />The yellow molecules are the only ones captured between <br />170V/cm-171V/cm in Cell #2 and further concentrated <br />And or separated before MS-detection.<br />Any Voltage fractions can be selected for targeted “Compound Fishing” experiments with 2D-EC NeedleInTheHaystack.<br />Capturing the essence of life<br />
  47. 47. +<br />-<br />Hydrodynamic flow<br /> Mass Spec<br />Electric Field<br />Ligand screening using Solution Phase Ion Mobility<br />Inject Ligand Cocktail<br />Compounds elute in order of increasing affinity<br />Using the protein as an “immobilised stationary phase” <br />
  48. 48. Conductive<br />membrane<br />125 m<br />Flow<br />Peek tubing<br />Captured protein<br />Image downstreamcathodejunctionduringcapture-deviceoperation<br />Protein captured at 30 nL spot<br />
  49. 49. rt 7.5 min<br />BSA<br />BSA<br />after<br />release of voltage<br />SB00396<br />
  50. 50. CompoundFishing-EpitopeImprinting<br />
  51. 51. The principle on the formation of MIP phase<br />Template = our handle<br />Mixing a template corresponding to the analyte/handle of interest with a compound (functional monomer) having the optimal bonding sites for the formation of hydrogen donor – acceptor interaction.<br />The functional monomer easily form at polymer in the presence of the template/handle, affording the correct cavity and bonding properties to the handle. Afterwards the template is removed.<br />This is analogous to a key in a lock. <br />
  52. 52. Principle of “our” MIP cavity bonding<br />The cavity match the functional group<br />The group has strong interaction due to hydrogen bonding.<br />X = reactive group that depending on its nature can “selectively” form a covalent bond to a functional group of the analyte of interest. Compound fishing .<br />
  53. 53. Compound fishing<br />If the analyte of interest has a functional group such as following examples:<br /><ul><li> -COOH
  54. 54. -NH2
  55. 55. -CO-
  56. 56. and many more</li></ul>The X can be selected to form a covalent bond with the particular group of<br /> the analyte, as shown in following example.<br />
  57. 57. What is special with our approach?<br /><ul><li>So far every analyte of interest most often require the formation of a dedicated MIP phase.
  58. 58. Our approach is generic, only one MIP phase is needed to capture almost </li></ul> any targeted organic compound . <br /><ul><li> We can selectively pick a compound or a group of compounds through </li></ul>derivatisation.<br /><ul><li>The MIP phase can be employed as a packed column for LC-MS, or</li></ul>on a surface using MALDI-MS.<br /><ul><li> MIP as biosensor (QCM, etc.).</li></li></ul><li>Beneficial….<br /><ul><li>Open up completely new diagnostic tool to analyze “stuff” not being possible before.
  59. 59. In addition analyze biomarkers that could not be monitored before at the required low levels.
  60. 60. New dimension for medical diagnostic purposes.
  61. 61. For a patients, both urine- and blood- samples can be analysed.
  62. 62. Urine samples by direct derivatisation.
  63. 63. Blood samples, by a simple purification step such as ultra</li></ul> filtration removing the proteins followed by derivatisation.<br />
  64. 64. Fee For Service<br />H/D Exchange<br />EpitopeMapping<br />Patent filingcases<br />ElectroCapturepre-concentration<br />Target is all Pharma and Biotech companiesworking with Ab, Proteins, Peptides etc.<br />
  65. 65. THANK YOU!<br />Thorleif Lavold CEO, Biomotif AB<br />tl@biomotif.com<br />www.biomotif.com<br />
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