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

Patent metadata

Field	Value
Publication number	US-2016349273-A1
Application number	US-201414902754-A
Country	US
Kind code	A1
Filing date	Jun 25, 2014
Priority date	Jul 12, 2013
Publication date	Dec 1, 2016
Grant date	—

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

1 . A method for the quantitative characterization of amyloid and/or aggregating peptides and/or proteins in a sample, comprising the steps of:
1. providing a sample, wherein the sample includes an amyloid and/or aggregating peptide and/or protein having at least one aggregate size and shape; 2. adding an active ingredient to be analyzed to the sample solution; 3. separating the amyloid and/or aggregating peptides and/or proteins from one another according to their aggregate size and shape; and 4. determining the change in concentration of the peptide and/or protein building blocks in at least one fraction by a comparison against control values without the active ingredient.

2 . The method according to claim 1 , wherein the amyloid and/or aggregated peptides and/or proteins of a particular fraction are denatured into monomer building blocks.

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

4 . The method according to claim 1 , wherein the preparation of a sample which contains as many as possible of those aggregates for which the change is to be analyzed.

5 . The method according to claim 1 , wherein the density gradient centrifugation takes place as fractionation.

6 . The method according to claim 1 , wherein the fractionation, in particular by a calibration of a density gradient centrifugation.

7 . The method according to claim 1 , wherein the concentration of the amyloid and/or aggregated peptides and/or proteins is determined by reverse phase (RP-) HPLC.

8 . The method according to claim 1 , wherein the denaturing of the amyloid and/or aggregated peptides and/or proteins into monomers is brought about at the same time, by way of the choice of mobile phase and/or by the choice of column temperature of the reverse phase (RP-) HPLC.

9 . The method according to claim 1 , wherein a mobile phase for the reverse phase HPLC is chosen which also enables the separation of the amyloid and/or aggregated peptides and/or proteins from the substance forming the density gradient.

10 . The method according to claim 1 , wherein during the method, a sample is provided in which aggregating Aβ, for example Aβ(1-40), Aβ(1-42), Aβ(1-43), Aβ(3-40), pyroGluAβ(3-40) and pyroGluAβ(3-42), or tau protein or another peptide, which typically occurs in Alzheimer's dementia and which has a high tendency to aggregate, is selected.

11 . The method according to claim 1 , wherein the active ingredient on the shape distribution of the amyloid and/or aggregated peptides and/or proteins in at least one fraction is also analyzed.

12 . The method according to claim 11 , wherein by analysis of the fractions by means of force field microscopy or electron microscopy or the like.

13 . The method according to claim 1 , wherein the change in concentration of peptide and/or protein building blocks in multiple fractions of the sample are compared with one another.

14 . The method according to claim 13 , wherein by comparison which determines the change in concentration of monomers and/or oligomers and/or fibrils and/or other conformers of a fraction under the effect of the active ingredient and relates this to other fractions.

15 . The method according to claim 1 , wherein a suitable active ingredient candidate for the purpose of treating a disease, based on a protein misfolding disease, such as Alzheimer's dementia for example, is provided, which allows few toxic molecules to form after being added.

16 . 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 multiple, at least 2, preferably at least 3, particularly preferably at least 4, 5, 6, 7, 8, 9, 10, 11, 0.12, 13, 14 or 15 fractions which can be analyzed.

17 . The method according to claim 1 , wherein after adding an active ingredient, the change in concentration of the peptide and/or protein building blocks in at least one fraction, preferably in at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or in all fractions which can be analyzed is determined by comparison against, control values without the active ingredient.

18 . The method according to claim 1 , wherein those fractions which, prior to the separation, also contained aggregating and/or aggregated peptides and/or proteins are included.

Assignees

• Forschungszentrum Juelich Gmbh

Inventors

• Nagel_Steger Luitgard
• Brener Oleksandr
• Gremer Lothar
• Willbold Dieter

Classifications

• G01N2333/4709

  Amyloid plaque core protein · CPC title

• B01D15/325

  Reversed phase · CPC title

• G01N2800/2821

  Alzheimer · CPC title

• G01N30/89

  Inverse chromatography · CPC title

• G01N33/6896 Primary

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