A new HX-IA Instrument for Mass Spectrometry. H/D Exchange without pipetting and NO dilution of sample. Molecular interactions, Epitope Mapping, Findtheneedleinthehaystack with On-line pI separation of proteins.

1. Capturing the essence of life HX-IA Instrument™ ThorleifLavold Biomotif AB

2. The HX-IA Instrument™ H/D Exchange to studyconformation, bindingsites, EpitopeMapping etc. ElectroCapture technology ”Findtheneedleinthehaystack” for on-line pI separation of proteins Ligand Screening CompoundFishing-EpitopeImprinting

4. New HX-IA Instrument

5. D2O H’s & D’s at backbone amide positions HDX Amide Hydrogen Exchange

6. −D −D −D −D −D −D −D −D −D −D −D −D −D −D −D −D −D −D −D −D −D −D −D −D −D −D −D −D −D −D −D −D H/D Exchange for Monitoring Protein Structural Changes IN HEAVY WATER Intensity FOLDED m/z mass shift After 10 min more D incorporated IN HEAVY WATER Intensity UNFOLDED m/z

8. Protein folding &

9. Conformational changes

10. Localisation of:

11. Epitope

12. Mutation

13. Aggregation

14. Ligand binding site

15. Complementary information to X-ray and NMRvs Folding Dynamics ? Conformational Changes Epitope Mapping Mutation Aggregation ? Ligand Binding ? ? Relation to X-Ray, NMR

17. Membrane –AssistedSample Preparation in the HX-IA Instrument Release/loop loading TrypsincColumnwash. LC-MS analysis Electrolytefluidiclines Buffer and waste bottles 50 mM NH4CH3COO 100% D2O 10 mM NH4CH3COO Gradient Pumps D2O D2O D2O Deuteration Isocratic Pump Autosampler Srynge Pumps Power supply 25 mM NH4CH3COO 1 % Formic Ac. Isocratic Pump Pepsin column Columnwashing Valve A Valve B SampleElution Gradient 5-95% ACN in 45min 0.1%FA Pre-column Loop Loading Mass Spectrometer Analytical Column Solvent lines 0.05 % TFA Waste lines

18. Membrane-Assisted Sample Preparation Semipermeable membrane separates sample channel from the deuterating solution Sample Channel Flow Semipermeable Membrane Second Solution Flow Semi-permeable Membranes

19. Membrane-Assisted Sample Preparation Semipermeable membrane separates the sample channel from the deuterating solution Sample Channel Flow Membrane Second Solution

20. Amyloid β Protein Monomer HDX

21. Different Deuteration LevelsAccording to Protein Conformation 15 % Deuteration PROTECTED 60 % Deuteration Unprotected 0 % Deuteration PROTECTED (Internal Pocket) 35 % Deuteration Semi-protected

22. Different Deuteration LevelsAccording to Protein Conformation

23. INTERLEIKIN 1β / ANTIBOBY H/D EXCHANGE EXPERIMENTS

24. Where does it bind? Epitope mapping by MS using 1H2H exchange (HDX) or covalent modification Unmodified Epitope Denature Digestion Surface Modification Protein Intact Mass MS Ligand Peptide Analysis DM Literature example of irreversible oxidative modification of Myoglobin

26. Flow Injection Deuteration (no need for automatedpipetting stations)

27. No need of liquid Nitrogen (online )

28. Acidification on-line with no dilution of sample

29. Tris or Phosphatebuffercan be used

32. Electrocapture-based Separations Positive Electrode Negative Electrode Charge particle Electrocapture conditions will be fulfilled when Ve≥ Vf Ve = Electrophoretic velocity ue = Electrophoresis mobility E = Electric field Electrophoretic velocity is given by, Ve= ue x E Capturing the essence of life

34. New Flat membrane cell

35. New cell in PEEK

36. AnalyticalApplications Capillaryelectrophoresis Protein band Flow MALDI & ESI-MS Micro reactions Anodic chamber Cathodic chamber Separations, μ-LC

37. Conductive membrane 125 m Flow Peek tubing Captured protein Image downstream cathode junction during capture-device operation Protein captured at 30 nL spot

38. ESI Total Ion Count of an Electrocapture-concentration Experiment 80-fold magnification! .

39. Desalting of BSA trypticpeptides MALDI-MS Astorga-Wells, J., Jörnvall, H. and Bergman, T. Anal. Chem.,2003, 75,5213-5219

40. Detergent Cleanup MALDI-MS BSA trypticpeptides Astorga-Wells, J., Jörnvall, H. and Bergman, T. Anal. Chem.,2003, 75,5213-5219

41. ESI-MS Analysis of α-Lactalbumin in a solution containing 0.5% Non-Ionic Detergent Detergent clusters n-octyl β-D glucopyranoside

42. ESI-MS Analysis of α-Lactalbumin in 0.5% Non-Ionic Detergent after Electrocapture Protein peaks After Electrocapture

43. Vf>Ve = ue x E Vf=Ve = ue x E Flow 200 E (V/cm) Time Capturing the essence of life

44. Electrocapture-based Separations of Proteins Beta-lactoglobulin 60 V Beta-casein 90 V Ribonuclease 120 V

45. FindtheneedleinthehaystackInstrument™

46. 2D-EC “NeedleInTheHaystack” Green, grey and purple fractions above 171 V/cm goes to waste + + - - Cell #1 is held at 170V/cm Cell #2 at 171V/cm The yellow molecules are the only ones captured between 170V/cm-171V/cm in Cell #2 and further concentrated And or separated before MS-detection. Any Voltage fractions can be selected for targeted “Compound Fishing” experiments with 2D-EC NeedleInTheHaystack. Capturing the essence of life

47. + - Hydrodynamic flow Mass Spec Electric Field Ligand screening using Solution Phase Ion Mobility Inject Ligand Cocktail Compounds elute in order of increasing affinity Using the protein as an “immobilised stationary phase”

48. Conductive membrane 125 m Flow Peek tubing Captured protein Image downstreamcathodejunctionduringcapture-deviceoperation Protein captured at 30 nL spot

49. rt 7.5 min BSA BSA after release of voltage SB00396

50. CompoundFishing-EpitopeImprinting

51. The principle on the formation of MIP phase Template = our handle 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. 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. This is analogous to a key in a lock.

52. Principle of “our” MIP cavity bonding The cavity match the functional group The group has strong interaction due to hydrogen bonding. 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 .

54. -NH2

55. -CO-

56. and many moreThe X can be selected to form a covalent bond with the particular group of the analyte, as shown in following example.

59. In addition analyze biomarkers that could not be monitored before at the required low levels.

60. New dimension for medical diagnostic purposes.

61. For a patients, both urine- and blood- samples can be analysed.

62. Urine samples by direct derivatisation.

63. Blood samples, by a simple purification step such as ultra filtration removing the proteins followed by derivatisation.

64. Fee For Service H/D Exchange EpitopeMapping Patent filingcases ElectroCapturepre-concentration Target is all Pharma and Biotech companiesworking with Ab, Proteins, Peptides etc.

65. THANK YOU! Thorleif Lavold CEO, Biomotif AB tl@biomotif.com www.biomotif.com