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.
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 technology<br />”Findtheneedleinthehaystack” for on-line pI separation of proteins <br />Ligand Screening<br />CompoundFishing-EpitopeImprinting<br />
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 />
Conclusions<br />Online Membrane-Assisted HDX<br />No D2O Dilution No Pippetting<br /><ul><li> Less sample consumption
Flow Injection Deuteration (no need for automatedpipetting stations)
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 />
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 />
+<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 />
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 />
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 />
Compound fishing<br />If the analyte of interest has a functional group such as following examples:<br /><ul><li> -COOH
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 />
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.
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.
In addition analyze biomarkers that could not be monitored before at the required low levels.
New dimension for medical diagnostic purposes.
For a patients, both urine- and blood- samples can be analysed.
Blood samples, by a simple purification step such as ultra</li></ul> filtration removing the proteins followed by derivatisation.<br />
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 />