Methods for carbon-centered radical mediated heavy hydrogen labeling of compounds
AssigneeCase Western Reserve University
InventorsVernon E. Anderson, Michael B. Goshe
Life of this patent
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Abstract
The solvent accessible reduced carbon atoms in a molecule of interest are labeled with heavy hydrogen. The carbon atoms are labeled under anaerobic, aqueous conditions by exposing the molecule of interest to a hydrogen atom abstractor in the presence of a heavy hydrogen source and a heavy hydrogen donor. If the hydrogen atom abstractor is hydroxyl radical, an electron scavenger source is provided. Optionally, an internal reference is provided to facilitate experimental reproducibility.
Claims67
A method of labeling a percentage of the solvent-accessible, reduced carbon atoms in a peptide or protein with heavy hydrogen, the method comprising:
- (i) generating hydroxyl radical in a solution for a time and under conditions effective to achieve a total equivalent concentration of hydroxyl radical of at least 10 μM, wherein said solution comprises the peptide or protein, an electron scavenger source, a heavy hydrogen source, a heavy hydrogen donor, and an oxygen trapping system, wherein the oxygen trapping system optionally comprises catalase;
- and
- (ii) adding an amount of a heavy hydrogen donor to the solution to replace heavy hydrogen donor that is depleted during step (i);
- wherein, when the total equivalent concentration of hydroxyl radical is achieved in step (i), the concentration of O2 dissolved in the solution is sufficiently low that a substantial number of the carbon-centered radicals formed in step (i) are repaired by donation of a hydrogen isotope by the heavy hydrogen donor.
A method of labeling a solvent-accessible, reduced carbon atom in a peptide or protein with deuterium, the method comprising generating a total equivalent concentration of hydroxyl radical of at least 10 μM in an aqueous N2O saturated solution that includes at least 50 picomoles of the peptide or protein, a molar ratio of D2O to total solution of at least 0.1 percent, at least 5 μM reduced dithiothreitol and an oxygen trapping system, the oxygen trapping system optionally comprising catalase, for a time and under conditions effective to label said solvent-accessible, reduced carbon atom;
- wherein the hydrogen atom abstractor reacts with the solvent-accessible, reduced carbon atom in the peptide or protein to form a corresponding carbon-centered radical of the solvent-accessible, reduced carbon atom and the reduced dithiothreitol donates the deuterium to the carbon-centered radical thereby labeling the solvent-accessible, reduced carbon atom in the peptide or protein with the deuterium;
- with the proviso that when the total equivalent concentration of hydroxyl radical is generated, the solution has an O2 concentration of less than 6 μM.
A method of labeling a solvent-accessible, reduced carbon atom in a peptide or protein with deuterium, the method comprising:
- (i) irradiating an N2O saturated aqueous solution with a 137Cs γ-ray source for a period of time sufficient to generate a total equivalent concentration of hydroxyl radical of at least 10 μM, the solution comprising at least 50 picomoles of the peptide or protein, a molar ratio of D2O to total solution of at least 0.1 percent, at least 5 μM reduced dithiothreitol, and an oxygen trapping system, wherein the oxygen trap system optionally comprises catalase;
- (ii) adding an amount of reduced, water-soluble dithiothreitol to the solution to replace reduced dithiothreitol depleted in step (i);
- (iii) contacting the solution with N2O gas, having less than 3000 parts per million O2, for at least five minutes;
- and
- (iv) repeating steps (i) thru (iii) until a cumulative total equivalent concentration of hydroxyl radical of 10 μM to 10 mM is achieved in the N2O saturated aqueous solution thereby labeling said solvent-accessible, reduced carbon atom.
A method of determining the amount of deuterium which has been incorporated into an amino acid of a peptide or protein comprising:
- (a) labeling a solvent-accessible, reduced carbon atom in a peptide or protein with deuterium by generating a hydrogen atom abstractor in a solution comprising the peptide or protein, a heavy hydrogen source, a heavy hydrogen donor, and an oxygen trapping system, for a time and under conditions effective to label a solvent-accessible, reduced carbon atom on said peptide or protein, wherein the solution is substantially oxygen free when the hydrogen atom abstractor is generated and wherein said oxygen trapping system optionally comprises catalase;
- (b) sequentially hydrolyzing the peptide or protein into a plurality of hydrolyzed amino acids after the hydrogen atom abstractor is generated;
- (c) optionally, derivatizing the plurality of hydrolized amino acids prior to purification;
- (d) purifying a sequentially hydrolyzed amino acid selected from the plurality of hydrolyzed amino acids;
- and
- (e) quantifying the amount of deuterium covalently bonded to a solvent-accessible, reduced carbon atom of the sequentially hydrolyzed amino acid.
A method of labeling a solvent-accessible, reduced carbon atom in a peptide or protein with a deuterium atom, the method comprising achieving a total equivalent concentration of hydroxyl radical of at least 10 μM in an aqueous N2O saturated solution comprising at least 50 picomoles of the peptide or protein, a molar ratio of D2O to total solution of at least 0.1 percent, at least 5 μM reduced dithiothreitol, and an oxygen trapping system, the oxygen trapping system optionally comprising catalase, for a time and under conditions effective to label said solvent-accessible, reduced carbon atom;
- wherein hydroxyl radical reacts with the solvent-accessible, reduced carbon atom in the peptide or protein to form a corresponding carbon-centered radical of the solvent-accessible, reduced carbon atom and the reduced dithiothreitol donates the deuterium to the carbon-centered radical thereby labeling the solvent-accessible reduced carbon atom in the peptide or protein with the deuterium;
- with the proviso that when the total equivalent concentration of hydroxyl radical is achieved, the aqueous N2O saturated solution has an O2 concentration of less than 6 μM.
A method of labeling a solvent-accessible, reduced carbon atom in a peptide or protein with deuterium, the method comprising:
- (i) irradiating an N2O saturated aqueous solution with a 137Cs γ-ray source for a period of time sufficient to generate a total equivalent concentration of hydroxyl radical of at least 10 μM, the solution comprising at least 50 picomoles of the peptide or protein, a molar ratio of D2O to total solution of at least 0.1 percent, at least 5 μM reduced dithiothreitol, and an oxygen trapping system, wherein said oxygen trapping system optionally comprises catalase;
- (ii) adding an amount of reduced, water-soluble dithiothreitol to the solution to replace reduced dithiothreitol depleted in step (i);
- (iii) contacting the solution with N2O gas, having less than 3000 parts per million O2, for at least five minutes;
- and
- (iv) repeating steps (i) thru (iii), under conditions effective to label said solvent-accessible, reduced carbon atom, until a cumulative total equivalent concentration of hydroxyl radical of 10 μM to 10 mM is generated in the N2O saturated aqueous solution.
A method of labeling solvent-accessible, reduced carbon atoms in a peptide or protein, the method comprising:
- generating a hydrogen atom abstractor in a solution comprising the peptide or protein, a heavy hydrogen source, a heavy hydrogen donor, and an oxygen trapping system, for a time and under conditions effective to label said solvent-accessible, reduced carbon atoms;
- wherein:
- (i) the solution is substantially oxygen free when the hydrogen atom abstractor is generated in the solution;
- (ii) the heavy hydrogen donor is selected from the group consisting of a reduced water soluble thiol, H2S, L-Ascorbic Acid, (±)-α-tocopherol, a phenol, a water soluble phosphine, and a water soluble phosphite;
- (iii) the hydrogen atom abstractor reacts, during said generating step, with said solvent-accessible, reduced carbon atoms to form corresponding carbon-centered radicals and the heavy hydrogen donor donates a heavy hydrogen to each said corresponding carbon-centered radical;
- and
- (iv) said oxygen trapping system optionally comprises catalase;
- with the proviso that when the heavy hydrogen donor is a water soluble phosphine or water soluble phosphite, the heavy hydrogen donor contains a bond selected from the group consisting of P—H, P—D, and P—T.
A method of labeling a solvent-accessible, reduced carbon atoms in a peptide or protein with deuterium, the method comprising:
- generating a hydrogen atom abstractor in a substantially oxygen free solution comprising the peptide or protein, a deuterium source, a heavy hydrogen donor, and an oxygen trapping system, for a time and under conditions effective to label said solvent-accessible, reduced carbon atoms to form corresponding carbon-centered radicals and the heavy hydrogen donor donating a deuterium to each said corresponding carbon-centered radical, wherein said oxygen trapping system optionally comprises catalase.
The method of 26, wherein the hydrogen atom abstractor is hydroxyl radical, and the solution includes an electron scavenger source when the total equivalent concentration of hydroxyl radical is generated.
A method of labeling a solvent-accessible, reduced carbon atom in a peptide or protein, the method comprising:
- generating a hydrogen atom abstractor in a substantially oxygen free solution comprising the peptide or protein, a heavy hydrogen source, a heavy hydrogen donor, an electron scavenger source, and an oxygen trapping system, for a time and under conditions effective to label said solvent-accessible, reduced carbon atom, the hydrogen atom abstractor reacting with said solvent-accessible, reduced carbon atom to form the corresponding carbon-centered radical with the heavy hydrogen donor donating a heavy hydrogen to said corresponding carbon-centered radical, wherein said oxygen trapping system optionally comprises catalase.
A method of labeling a percentage of the solvent-accessible, reduced carbon atoms in a peptide or protein with heavy hydrogen, the method comprising:
- (i) generating hydroxyl radical in a solution for a time and under conditions effective to achieve a total equivalent concentration of hydroxyl radical of at least 10 μM, wherein said solution comprises the peptide or protein, an electron scavenger source, a heavy hydrogen source, a heavy hydrogen donor, and an oxygen trapping system, wherein the oxygen trapping system optionally comprises catalase;
- and
- (ii) adding an amount of a heavy hydrogen donor to the solution to replace heavy hydrogen donor that is depleted during step (i);
- and
- (iii) optionally repeating steps (i) and (ii) until a cumulative total equivalent concentration of hydroxyl radical generated in the solution is sufficient to produce carbon-centered radicals of said percentage of solvent-accessible, reduced carbon atoms in said peptide or protein;
- wherein, when the total equivalent concentration of hydroxyl radical is achieved in step (i), the concentration of O2 dissolved in the solution is sufficiently low that a substantial number of the carbon-centered radicals formed in step (i) are repaired by donation of a hydrogen isotope by the heavy hydrogen donor.
Dependencies are inferred from the claim text.