Method for the quantitative characterization of amyloid and/or aggregating peptides and/or proteins in a sample

Patent metadata

Field	Value
Publication number	US-10088487-B2
Application number	US-201414902754-A
Country	US
Kind code	B2
Filing date	Jun 25, 2014
Priority date	Jul 12, 2013
Publication date	Oct 2, 2018
Grant date	Oct 2, 2018

Abstract

A method for the quantitative characterization of amyloid and/or aggregated peptides and/or proteins in a sample, comprising:—providing a sample, wherein the sample includes an amyloid and/or aggregated peptide and/or protein having at least one aggregate size and shape;—adding an active ingredient to be analyzed to the sample solution;—separating the amyloid and/or aggregated peptides and/or proteins are from one another according to their aggregate size and shape;—optionally completely denaturing the amyloid and/or aggregated peptides and/or proteins of a particular fraction into monomer building blocks;—determining the change in concentration of the peptide and/or protein building blocks in at least one fraction by comparison against control values without the active ingredient.

First claim

The invention claimed is:

1. A method for quantitatively determining an effect of an active ingredient on a concentration of amyloid and/or aggregating peptides and/or proteins in a sample solution, comprising the steps of:
providing a sample in the sample solution, wherein the sample includes said amyloid and/or aggregating peptide and/or proteins having at least one aggregate size and shape;
adding said active ingredient to the sample solution;
separating the amyloid and/or aggregating peptides and/or proteins from one another according to their aggregate size and shape by density gradient centrifugation to achieve fractionation of the sample into a plurality of fractions;
completely denaturing the amyloid and/or aggregated peptides and/or proteins of a first fraction among said plurality of fractions into monomer building blocks; and
after said completely denaturing, determining a change in concentration of peptides and/or proteins among the amyloid and/or aggregated peptides and/or proteins of said first fraction by a comparison against control values without the active ingredient.

2. The method according to claim 1 wherein multiple active ingredients are tested in a screening process in vitro.

3. The method according to claim 1 , wherein said provided sample contains a plurality of aggregate sizes which become separated into said plurality of fractions, and said determining step comprising determining said change in concentration for each one fraction of said plurality of fractions.

4. The method according to claim 1 , wherein said separating comprises performing said density gradient centrifugation with calibration to obtain fractionation of the prepared sample.

5. The method according to claim 1 , wherein said density gradient centrifugation is calibrated, according to a sedimentation coefficient, for performing said separating.

6. The method according to claim 1 , wherein said determining comprises determining using reverse phase (RP-) HPLC.

7. The method according to claim 6 , wherein said completely denaturing occurs during the reverse phase (RP-) HPLC, prior to said determining said change in concentration, the amyloid and/or aggregated peptides and/or proteins being completely denatured into monomers based upon a choice of mobile phase and/or a choice of column temperature of the reverse phase (RP-) HPLC.

8. The method according to claim 1 , wherein upon said separating said plurality of fractions are situated according to a density gradient, and further comprising performing a reverse phase (RP-) HPLC, wherein a mobile phase for said reverse phase (RP-) HPLC is chosen which provides separation of the amyloid and/or aggregated peptides and/or proteins for each fraction of said plurality of fractions of said density gradient.

9. The method according to claim 1 , wherein during the method, a sample is provided in which an aggregating Aβ is included from among a group comprising Aβ(1-40), Aβ(1-42), Aβ(1-43), Aβ(3-40), pyroGluAβ(3-40), pyroGluAβ(3-42), tau protein or another peptide typically occurring in Alzheimer's dementia and having a high tendency to aggregate.

10. The method according to claim 1 , further comprising, after said separating, determining a change in shape distribution of the amyloid and/or aggregated peptides and/or proteins in said first fraction as an effect of said active ingredient.

11. The method according to claim 10 , wherein said change in shape distribution is determined using force field microscopy or electron microscopy or the like.

12. The method according to claim 1 , wherein said provided sample includes said amyloid and/or aggregating peptide and/or proteins having a plurality of aggregate sizes and shapes;
wherein said completely denaturing comprises completely denaturing the amyloid and/or aggregated peptides and/or proteins of each one fraction of multiple fractions among said plurality of fractions into monomer building blocks; and
wherein said determining comprises determining a change in concentration of peptides and/or proteins among the amyloid and/or aggregated peptides and/or proteins of each one fraction of said multiple fractions by a comparison against control values without the active ingredient.

13. The method according to claim 12 , wherein said determining said change in concentration comprises determining the change in concentration of monomers and/or oligomers and/or fibrils and/or other conformers of each one fraction of said multiple fractions under the effect of the active ingredient.

14. The method according to claim 1 , wherein said active ingredient has an effect on said provided sample in said sample solution of minimizing toxic molecules formed so as to treat a protein mis-folding disease.

15. The method according to claim 1 , wherein the amyloid and/or aggregated peptides and/or proteins are separated from one another according to aggregate size and shape to form 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 fractions which can be analyzed.

16. The method according to claim 1 , wherein said determining comprises determining said change in concentration of peptides and/or proteins among the amyloid and/or aggregated peptides and/or proteins of multiple fractions among said plurality of fractions by comparison against control values without the active ingredient.

17. The method according to claim 1 , wherein a concentration of peptides and/or proteins among the amyloid and/or aggregated peptides and/or proteins prior to said separating is known, and further comprising deriving a recovery rate for said peptides and/or proteins among the amyloid and/or aggregated peptides and/or proteins by comparing the concentrations known prior to said separating to the concentrations determined in said determining step.

18. The method according to claim 1 , wherein said determining the change in concentration is performed for each one fraction of the plurality of fractions, the change in concentration within each one fraction of the plurality of fractions being based on a comparison against at least one control sample in sample solution without the active ingredient.

19. The method according to claim 1 ,
wherein each one fraction of said plurality of fractions of the sample resulting from said density gradient centrifugation is characterized by an aggregation state of the the amyloid and/or aggregating peptides and/or proteins; and
wherein said completely denaturing is achieved by reverse phase (RP-) HPLC for each one fraction of the plurality of fractions, said reverse phase (RP-) HPLC being effective to completely denature into monomers the amyloid and/or aggregated peptides and/or proteins of each one fraction of the plurality of fractions, regardless of the aggregation state, based upon a choice of mobile phase and/or a choice of column temperature of the reverse phase (RP-) HPLC.

Assignees

• Forschungszentrum Juelich Gmbh

Inventors

• Nagel-Steger Luitgard
• Brener Oleksandr
• Gremer Lothar
• Willbold Dieter

Classifications

• B01D15/325

  Reversed phase · CPC title

• G01N33/6896 Primary

  Neurological disorders, e.g. Alzheimer's disease · CPC title

• G01N33/6845 Primary

  Methods of identifying protein-protein interactions in protein mixtures · CPC title

• G01N2500/00

  Screening for compounds of potential therapeutic value · CPC title

• G01N30/89

  Inverse chromatography · CPC title