Detection of conformationally altered proteins
The invention provides methods and kits for detecting conformationally altered proteins and prions in a sample. In one embodiment, the conformationally altered proteins and prions are associated with amyloidogenic diseases.
1. A method for detecting misfolded Aβ peptide in a sample comprising:
(a) contacting a liquid sample that comprises a liquid biological sample obtained from a subject and does not contain homogenized tissue with a peptide probe comprising an amino acid sequence corresponding to a β-sheet forming region of said Aβ peptide, wherein the peptide probe exhibits a random coil or alpha-helix conformation in solution and undergoes a transition to a β-sheet conformation upon interaction with Aβ peptide exhibiting a β-sheet conformation, wherein the amino acid sequence of the peptide probe comprises a sequence that is at least 70% identical to any one of SEQ ID NOs: 4-7,
(b) permitting the peptide probe to interact with any misfolded Aβ peptide present in the sample; and
(c) detecting any interaction between the peptide probe and any misfolded Aβ peptide present in the sample.
2. The method of claim 1 , wherein the peptide probe is labeled with a detectable label.
3. The method of claim 1 , wherein
(i) both termini of the peptide probe are labeled with a fluorophore capable of forming excimers and
(ii) the detecting step comprises detecting any excimers formed upon interaction between the fluorophore-labeled peptide probe and any Aβ peptide present in the sample.
4. The method of claim 1 , wherein the detecting step comprises using circular dichroism to detect any interaction between the peptide probe and any Aβ peptide present in the sample.
5. The method of claim 1 , further comprising, prior to the contacting step, subjecting the sample to a disaggregation step.
6. The method of claim 1 , wherein the liquid sample comprises a liquid biological sample selected from the group consisting of blood, a blood fraction, plasma, serum, and spinal fluid.
7. The method of claim 1 , wherein the peptide probe comprises an amino acid sequence that is at least about 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs 4-7.
8. The method of claim 1 , wherein the peptide probe consists of from 10 to 50 amino acids.
9. The method of claim 1 , wherein the amino acid sequence corresponding to a β-sheet forming region of the Aβ peptide is at least about 70%, at least about 90% or 100% identical to the β-sheet forming region of the Aβ peptide.
10. The method of claim 2 , wherein the detectable label is selected from
(i) optically detectable moieties and
(ii) radionuclides.
11. The method of claim 10 , wherein the detectable label is a chromophore.
12. The method of claim 10 , wherein the detectable label is selected from pyrene, tryptophan, fluorescein, rhodamine, 1-anilino-8-napthalene sulfonate (ANS), Congo Red stain and fluorescence resonance energy transfer (FRET) labels.