Methods for determining molecule folding associated with proteopathies
Methods, systems, and compositions for detecting molecule aggregation, folding, or interactions featuring comparing the amount of labeling of a molecule of interest, such as a protein, in a test sample with an amount of labeling in a control, e.g., a sample wherein the molecule of interest is denatured. If less labeling is present in the test sample as compared to the control sample, the test sample may comprise the molecule of interest in aggregate form, folded form, or interactive form, e.g., interacting with another molecule such as a protein molecule, DNA molecule or RNA molecule. The present invention may be used for detecting or monitoring a disease or condition such as a protein misfolding disease (proteopathy), e.g., amyotrophic lateral sclerosis (ALS), etc.
1. A method of detecting the presence of occluded amino acids in a protein associated with a proteopathy in a test sample, said method comprising:
a. subjecting the test sample to a reaction adapted to covalently modify tyrosines with a first reactive moiety conjugated to a first detectable label;
b. subjecting the test sample to denaturing conditions after labeling the sample with the first reactive moiety conjugated to the first detectable label;
c. subjecting the test sample to a reaction adapted to covalently modify tyrosines with the first reactive moiety conjugated to a second detectable label after subjecting the sample to denaturing conditions, wherein the first detectable label and second detectable label are visually distinct; and
d. making visible the first detectable label and second detectable label;
wherein the presence of the second detectable label in addition to the first detectable label is indicative of the presence of occluded tyrosines in the protein associated with a proteopathy in the test sample.
2. The method of claim 1 , wherein the presence of occluded amino acids in the protein associated with a proteopathy in the test sample is indicative of a presence of a proteopathy.
3. The method of claim 1 , wherein the proteopathy is Alzheimer's disease, Parkinson's disease, Creutzfeldt-Jacob disease, prion disease, an amyloidosis, Huntington's disease, frontotemporal lobar degeneration (FTLD), type 2 diabetes, a cancer associated with p53 aggregation, amyotrophic lateral sclerosis (ALS), chronic traumatic encephalopathy, dementia, tauopathies, retinal ganglion cell degeneration, cerebral beta amyloid angiopathy, Alexander disease, cerebral hemorrhage with amyloidosis, CADASIL, seipinopathies, familial amyloidotic neuropathy, senile systemic amyloidosis, cataracts, medullary thyroid carcinoma, pituitary prolactinoma, cystic fibrosis, sickle cell disease, or pulmonary alveolar proteinosis.
4. The method of claim 1 , wherein the protein associated with a proteopathy is selected from fused in sarcoma (FUS), TDP-43, hnRNPA1, GRN, SQSTM1, SOD1, PFN1, VCP, OPTN, SETX, ANG, hnRNPA2B1, UBQLN2, APP, Tau, APPBP2, APCS, APBA2, PSEN1, PSEN2, HTT, Alpha-synuclein, NEFL light chain, NEFL medium chain, p53, IAPP, insulin, B2M, PrP, or a combination thereof.
5. The method of claim 1 , wherein the first detectable label or second detectable label comprises coumarin, fluorophores, radiolabels, heavy isotopes, metal chelators, biotin, peptides, fluorescent microspheres, fluorescent proteins, quantum dots, or a combination thereof.
6. The method of claim 1 , wherein occluded amino acids are associated with a protein in a bound state.
7. The method of claim 1 , wherein occluded amino acids are associated with a protein in a folded state.
8. The method of claim 1 , wherein occluded amino acids are associated with a protein in an interactive state wherein the protein interacts with a second molecule.
9. The method of claim 1 , wherein making visible the detectable label comprises subjecting the sample to fluorescence spectroscopy, imaging, NMR, chromatography, electrophoresis, affinity purification, immunopurification, or a combination thereof.
10. The method of claim 1 , wherein the first reactive moiety comprises 4-phenyl-3H-1,2,4-triazole-3,5(4H)-dione (PTAD) or a diazonium compound.
11. The method of claim 1 , wherein the reaction adapted to covalently modify tyrosines with the first reactive moiety conjugated to the first detectable label comprises 4-phenyl-3H-1,2,4-triazole-3,5(4H)-dione (PTAD) chemistry.
12. The method of claim 1 , wherein the reaction adapted to covalently modify tyrosines with the first reactive moiety conjugated to the first detectable label comprises diazonium chemistry.