Dyes for analysis of protein aggregation

Abstract

Provided are dyes and compositions which are useful in a number of applications, such as the detection and monitoring protein aggregation, kinetic studies of protein aggregation, neurofibrillary plaques analysis, evaluation of protein formulation stability, and analysis of molecular chaperone activity.

What is claimed is: 
     
         1 . A compound comprising the structure 
       
         
           
           
               
               
           
         
         wherein m and n are independently 1, 2 or 3; 
         wherein L is a linker arm comprising carbon, sulfur, oxygen, nitrogen, or any combination thereof; 
         wherein R 1 , R 2 , R 3 , R 4 , R 9 , R 10 , R 11 , R 12 , R 13 , R 14 , R 15 , R 16 , R 19 , R 20 , R 21  and R 22  are independently hydrogen, halogen, amino, ammonium, nitro, sulfo, sulfonamide, carboxy, ester, cyano, phenyl, benzyl, an alkyl group wherein the alkyl group is saturated or unsaturated, linear or branched, substituted or unsubstituted, an alkoxy group wherein the alkoxy group is saturated or unsaturated, branched or linear, substituted or unsubstituted, or when taken in combination R 1  and R 2 , or R 3  and R 4 , or R 9  and R 10 , or R 11  and R 12 , or R 13  and R 14 , or R 15  and R 16 , or R 19  and R 20 , or R 21  and R 22  form a five or six membered ring wherein the ring is saturated or unsaturated, substituted or unsubstituted, and wherein R 9  and R 10 , or R 11  and R 12 , or R 13  and R 14 , or R 15  and R 16  can comprise alkyl chains that are joined together, wherein a quinoline moiety can be formed; 
         wherein R 7 , R 8 , R 17  and R 18  are independently hydrogen, Z, an alkyl group wherein the alkyl group is saturated or unsaturated, linear or branched, substituted or unsubstituted, an alkoxy group wherein the alkoxy group is saturated or unsaturated, branched or linear, substituted or unsubstituted, or when taken together, R 7  and R 8  and R 17  and R 18 , may form a 5 or 6 membered ring wherein the ring is saturated or unsaturated, substituted or unsubstituted;
 wherein Z comprises a carboxyl group (CO 2   − ), a carbonate ester (COER 25 ), a sulfonate (SO 3   − ), a sulfonate ester (SO 2 ER 25 ), a sulfoxide (SOR 25 ), a sulfone (SO 2 CR 25 R 26 R 27 ), a sulfonamide (SO2NR 25 R 26 ), a phosphate (PO 4   = ), a phosphate monoester (PO 3   − ER 25 ), a phosphate diester (PO 2 ER 25 ER 26 ), a phosphonate (PO 3   = ) a phosphonate monoester (PO 2   − ER 25 ) a phosphonate diester (POER 25 ER 26 ), a thiophosphate (PSO 3   = ), a thiophosphate monoester (PSO 2   − ER 25 ) a thiophosphate diester (PSOER 25 ER 26 ), a thiophosphonate (PSO 2   = ), a thiophosphonate monoester (PSO − ER 25 ) a thiophosphonate diester (PSER 25 ER 26 ), a phosphonamide (PONR 25 R 26 NR 28 R 29 ), its thioanalogue (PSNR 25 R 26 NR 28 R 29 ), a phosphoramide (PONR 25 R 26 NR 27 NR 28 R 29 ), its thioanalogue (PSNR 25 R 26 NR 27 NR 28 R 29 ), a phosphoramidite (PO 2 R 25 NR 28 R 29 ) or its thioanalogue (POSR 25 NR 28 R 29 ) where E can be independently O or S;
 wherein R 25 , R 26 , R 27 , R 28 , and R 29  are independently a hydrogen, an unsubstituted straight-chain, branched or cyclic alkyl, alkenyl or alkynyl group, a substituted straight-chain, branched or cyclic alkyl, alkenyl or alkynyl group wherein one or more C, CH or CH 2  groups are substituted with an O atom, N atom, S atom, or NH group, or an unsubstituted or substituted aromatic group; 
 
 wherein Z is attached directly, or indirectly through a second linker arm comprising carbon, sulfur, oxygen, nitrogen, and any combinations thereof and wherein the second linker arm may be saturated or unsaturated, linear or branched, substituted or unsubstituted or any combinations thereof; and 
 
         wherein R 5 , R 6 , R 23  and R 24  can independently be hydrogen or an alkyl group wherein the alkyl group is saturated or unsaturated, linear or branched, substituted or unsubstituted, or when taken in combination R 5  and R 6  or R 2  and R 5  or R 3  and R 6  or R 23  and R 24  or R 22  and R 23  or R 20  and R 24  form a five or six membered ring wherein the ring is saturated or unsaturated, substituted or unsubstituted. 
       
     
     
         2 . The compound of  claim 1 , wherein the compound exhibits increased fluorescence in the presence of an aggregated form of a protein when compared to the fluorescence exhibited when the compound is in the presence of the unaggregated form of the protein. 
     
     
         3 . The compound of  claim 2 , wherein the protein is immunoglobulin, a DNA polymerase or a fragment thereof, α-synuclein, synphilin-1, TCRα, P23H mutant of rhodopsin, ΔF508 mutant of CFTR, amyloid-β, prion protein, Tau, SOD1, Ig light chains, ataxin-1, ataxin-3, ataxin-7, calcium channel, atrophin-1, androgen receptor, p62/sequestosome1 (SQSTM1), Pael receptor, serum amyloid A, transthyretin, β2-microglobulin, apolipoprotein A-1, gelsolin, atrial natriuretic factor, lysozyme, insulin, fibrinogen, crystallin, surfactant protein C, lactoferrin, βig-h3, PAPB2, corneodesmosin, neuroserpin, cochlin, RET, myelin, protein 22/0, SCAD, prolactin, lactadherin, p53, procalcitonin, cytokeratin, GFAP, ATP7B, prolyl hydroxylase PHD3, presenilin, or huntingtin. 
     
     
         4 . The compound of  claim 1 , comprising the structure 
       
         
           
           
               
               
           
         
       
     
     
         5 . The compound of  claim 4 , wherein each of R 5 , R 6 , R 23  and R 24  are a methyl or an ethyl moiety. 
     
     
         6 . The compound of  claim 1 , wherein the compound comprises S25, S43, TOL3, YAT2134, YAT2148, YAT2149, S13, YAT2135, YAT2324 or YAT2150. 
     
     
         7 . The compound of  claim 1 , wherein the compound is S25. 
     
     
         8 . The compound of  claim 1 , wherein the compound is S43. 
     
     
         9 . The compound of  claim 1 , wherein the compound is TOL3. 
     
     
         10 . The compound of  claim 1 , wherein the compound is YAT2134. 
     
     
         11 . The compound of  claim 1 , wherein the compound is YAT2148. 
     
     
         12 . The compound of  claim 1 , wherein the compound is YAT 2149. 
     
     
         13 . The compound of  claim 1 , wherein the compound is S13. 
     
     
         14 . The compound of  claim 1 , wherein the compound is YAT2135. 
     
     
         15 . The compound of  claim 1 , wherein the compound is YAT2324. 
     
     
         16 . The compound of  claim 1 , wherein the compound is YAT2150. 
     
     
         17 . A compound exhibiting at least three times increased fluorescence in the presence of an aggregated form of a protein when compared to the fluorescence exhibited when the compound is in the presence of the unaggregated form of the protein, wherein the compound is D95, D97, L-30, L-33, Lu-1, Lu-2, S-8, S13. S22, S25, S33, S39, S42, S43, S48, S49, SL2131, SL2592, Tio-1, TOL-2, TOL-3, TOL-5, TOL-6, TOL-7, TOL-11, YA-1, YA-3, YAT2134, YAT2135, YAT2148, YAT2149, YAT2150, YAT2213, YAT2214 or YAT2324. 
     
     
         18 . A multi-dye composition comprising at least three dyes, wherein each of the at least three dyes exhibits increased fluorescence in the presence of an aggregated form of a protein when compared to the fluorescence exhibited when the compound is in the presence of the unaggregated form of the protein. 
     
     
         19 . The multi-dye composition of  claim 18 , wherein, in the presence of the aggregated form of the protein, the emission maximum of each dye is within 150 nm of the emission maximum of each of the other dyes. 
     
     
         20 . The multi-dye composition of  claim 18 , wherein, in the presence of the aggregated form the protein, the emission maximum of each dye is within 50 nm of the emission maximum of each of the other dyes. 
     
     
         21 . The multi-dye composition of  claim 18 , wherein the protein is immunoglobulin, a DNA polymerase or a fragment thereof, α-synuclein, synphilin-1, TCRα, P23H mutant of rhodopsin, ΔF508 mutant of CFTR, amyloid-β, prion protein, Tau, SOD1, Ig light chains, ataxin-1, ataxin-3, ataxin-7, calcium channel, atrophin-1, androgen receptor, p62/sequestosome1 (SQSTM1), Pael receptor, serum amyloid A, transthyretin, β2-microglobulin, apolipoprotein A-1, gelsolin, atrial natriuretic factor, lysozyme, insulin, fibrinogen, crystallin, surfactant protein C, lactoferrin, βig-h3, PAPB2, corneodesmosin, neuroserpin, cochlin, RET, myelin, protein 22/0, SCAD, prolactin, lactadherin, p53, procalcitonin, cytokeratin, GFAP, ATP7B, prolyl hydroxylase PHD3, presenilin, or huntingtin. 
     
     
         22 . The multi-dye composition of  claim 18 , wherein each of the three dyes is selected from the group consisting of Dye F, Dye Fm(b), D95, D97, L-30, L-33, Lu-1, Lu-2, S-8, S13. S22, S25, S33, S39, S42, S43, S48, S49, SL2131, SL2592, Tio-1, TOL-2, TOL-3, TOL-5, TOL-6, TOL-7, TOL-11, YA-1, YA-3, YAT2134, YAT2135, YAT2148, YAT2149, YAT2150, YAT2213, YAT2214 and YAT2324. 
     
     
         23 . The multi-dye composition of  claim 18 , wherein at least one of the three dyes is selected from the group consisting of S25, S43, TOL3, YAT2134, YAT2148, YAT2149, S13, YAT2135, YAT2324 and YAT2150. 
     
     
         24 . A multi-dye composition comprising two or more dyes, wherein at least one of the two or more dyes comprises Dye F, Dye Fm(b), D95, D97, L-30, L-33, Lu-1, Lu-2, S-8, S13. S22, S25, S33, S39, S42, S43, S48, S49, SL2131, SL2592, Tio-1, TOL-2, TOL-3, TOL-5, TOL-6, TOL-7, TOL-11, YA-1, YA-3, YAT2134, YAT2135, YAT2148, YAT2149, YAT2150, YAT2213, YAT2214 or YAT2324. 
     
     
         25 . The multi-dye composition of  claim 24 , wherein, in the presence of an aggregated form of a protein, the emission maximum of each dye is within 150 nm of the emission maximum of each of the other dyes. 
     
     
         26 . The multi-dye composition of  claim 24 , wherein, in the presence of an aggregated form of a protein, the emission maximum of each dye is within 50 nm of the emission maximum of each of the other dyes. 
     
     
         27 . The multi-dye composition of  claim 24 , wherein at least one of the two dyes is selected from the group consisting of S25, S43, TOL3, YAT2134, YAT2148, YAT2149, S13, YAT2135, YAT2324 and YAT2150. 
     
     
         28 . The multi-dye composition of  claim 24 , wherein both of the two dyes are selected from the group consisting of S25, S43, TOL3, YAT2134, YAT2148, YAT2149, S13, YAT2135, YAT2324 and YAT2150. 
     
     
         29 . The multi-dye composition of  claim 24 , wherein the two dyes are S25 and TOL3. 
     
     
         30 . A reactive compound comprising at least one compound from Table 1B or Table 2B, wherein said compound is modified by the addition of a reactive group. 
     
     
         31 . The reactive compound of  claim 30 , wherein the reactive group comprises an electrophilic reactive group comprising an isocyanate, isothiocyanate, monochlorotriazine, dichlorotriazine, 4,6,-dichloro-1,3,5-triazines, mono- or di-halogen substituted pyridine, mono- or di-halogen substituted diazine, maleimide, haloacetamide, aziridine, sulfonyl halide, acid halide, hydroxysuccinimide ester, hydroxysulfosuccinimide ester, imido ester, hydrazine, azidonitrophenol, azide, 3-(2-pyridyl dithio)-propionamide, glyoxal or aldehyde group, or a combination thereof. 
     
     
         32 . The reactive compound of  claim 30 , wherein the reactive group comprises a nucleophilic reactive group comprising a reactive thiol, amine or hydroxyl, or a combination thereof. 
     
     
         33 . The reactive compound of  claim 30 , wherein the reactive group comprises a terminal alkene group, a terminal alkyne group, a nickel coordinate group or a platinum coordinate group. 
     
     
         34 . The reactive compound of  claim 30 , wherein the reactive group is attached to the compound through a linker arm. 
     
     
         35 . A labeled target molecule comprising a target molecule attached to the reactive compound of  claim 30  through the reactive group. 
     
     
         36 . The labeled target molecule of  claim 35 , wherein the target molecule comprises a nucleoside, nucleotide, oligonucleotide, polynucleotide, peptide nucleic acid, protein, peptide, enzyme, antigen, antibody, hormone, hormone receptor, cellular receptor, lymphokine, cytokine, hapten, lectin, avidin, streptavidin, digoxigenin, carbohydrate, oligosaccharide, polysaccharide, lipid, liposomes, glycolipid, viral particle, viral component, bacterial cell, bacterial component, eukaryotic cell, eukaryotic cell component, natural drug, synthetic drug, or a combination thereof. 
     
     
         37 . The labeled target molecule of  claim 21 , wherein a linker arm is between the compound and the reactive group. 
     
     
         38 . The labeled target molecule of  claim 21 , wherein a linker arm is between the target molecule and the reactive group. 
     
     
         39 . A solid support attached to the reactive compound of  claim 30  through the reactive group. 
     
     
         40 . The solid support of  claim 39 , comprising glass particles, glass surface, natural polymers, synthetic polymers, plastic particles, plastic surface, silicaceous particles, silicaceous surface, glass, plastic beads, latex beads, controlled pore glass, metal particles, metal oxide particles, a microplate, a microarray, or combinations thereof. 
     
     
         41 . The solid support of  claim 39 , wherein a linker arm is between the compound and the reactive group. 
     
     
         42 . The solid support of  claim 39 , wherein a linker arm is between the solid support and the reactive group. 
     
     
         43 . A kit for assaying aggregation of a protein, comprising in packaged combination:
 (a) the compound of  claim 17 , and   (b) instructions for using the compound for assaying aggregation of a protein.   
     
     
         44 . The kit of  claim 43 , further comprising a buffer, a positive control, a negative control, or a combination thereof. 
     
     
         45 . The kit of  claim 43 , further comprising a positive control of an aggregated protein and a negative control of an unaggregated protein. 
     
     
         46 . The kit of  claim 43 , further comprising a microplate or a microarray. 
     
     
         47 . The kit of  claim 43 , wherein the protein is immunoglobulin, a DNA polymerase or a fragment thereof, α-synuclein, synphilin-1, TCRα, P23H mutant of rhodopsin, ΔF508 mutant of CFTR, amyloid-β, prion protein, Tau, SOD1, Ig light chains, ataxin-1, ataxin-3, ataxin-7, calcium channel, atrophin-1, androgen receptor, p62/sequestosome1 (SQSTM1), Pael receptor, serum amyloid A, transthyretin, β2-microglobulin, apolipoprotein A-1, gelsolin, atrial natriuretic factor, lysozyme, insulin, fibrinogen, crystallin, surfactant protein C, lactoferrin, βig-h3, PAPB2, corneodesmosin, neuroserpin, cochlin, RET, myelin, protein 22/0, SCAD, prolactin, lactadherin, p53, procalcitonin, cytokeratin, GFAP, ATP7B, prolyl hydroxylase PHD3, presenilin, or huntingtin. 
     
     
         48 . A kit for assaying aggregation of a protein, comprising in packaged combination:
 (a) two or more compounds, wherein each of compound exhibits increased fluorescence in the presence of an aggregated form of a protein when compared to the fluorescence produced when the compound is in the presence of the unaggregated form of the protein, and   (b) instructions therefor.   
     
     
         49 . The kit of  claim 48 , wherein the emission maxima of the compounds is within 50 nanometers of each other when measured in the presence of the aggregated protein. 
     
     
         50 . The kit of  claim 48 , wherein the emission maxima of the compounds is within 10 nanometers of each other when measured in the presence of the aggregated protein. 
     
     
         51 . The kit of  claim 48 , wherein the emission maxima of the compounds is from about 600 nanometers to about 670 nanometers when measured in the presence of the aggregated protein. 
     
     
         52 . The kit of  claim 48 , further comprising a buffer, a positive control, a negative control, or a combination thereof. 
     
     
         53 . The kit of  claim 48 , further comprising a positive control of an aggregated protein and a negative control of an unaggregated protein. 
     
     
         54 . The kit of  claim 48 , wherein at least one of the compounds is the compound of  claim 2 . 
     
     
         55 . The kit of  claim 48 , wherein at least one of the compounds is Dye F, Dye Fm(b), D95, D97, L-30, L-33, Lu-1, Lu-2, S-8, S13. S22, S25, S33, S39, S42, S43, S48, S49, SL2131, SL2592, Tio-1, TOL-2, TOL-3, TOL-5, TOL-6, TOL-7, TOL-11, YA-1, YA-3, YAT2134, YAT2135, YAT2148, YAT2149, YAT2150, YAT2213, YAT2214 or YAT2324. 
     
     
         56 . The kit of  claim 48 , wherein the protein is immunoglobulin, a DNA polymerase or a fragment thereof, α-synuclein, synphilin-1, TCRα, P23H mutant of rhodopsin, ΔF508 mutant of CFTR, amyloid-β, prion protein, Tau, SOD1, Ig light chains, ataxin-1, ataxin-3, ataxin-7, calcium channel, atrophin-1, androgen receptor, p62/sequestosome1 (SQSTM1), Pael receptor, serum amyloid A, transthyretin, β2-microglobulin, apolipoprotein A-1, gelsolin, atrial natriuretic factor, lysozyme, insulin, fibrinogen, crystallin, surfactant protein C, lactoferrin, βig-h3, PAPB2, corneodesmosin, neuroserpin, cochlin, RET, myelin, protein 22/0, SCAD, prolactin, lactadherin, p53, procalcitonin, cytokeratin, GFAP, ATP7B, prolyl hydroxylase PHD3, presenilin, or huntingtin. 
     
     
         57 . A method for detecting an aggregate of a protein in a sample, the method comprising
 (a) combining the sample with the compound of  claim 2  or  17 , or the multidye composition of  claim 18  or  24  to form a dye-sample mixture;   (b) measuring the amount of fluorescence in the dye-sample mixture;   (c) comparing the amount of fluorescence determined in (b) with the amount of fluorescence in
 (i) a mixture of the compound or multidye composition with a control sample without aggregated protein, or 
 (ii) a mixture of the compound or multidye composition with a known standard quantity of aggregated protein; and 
   (d) determining the aggregation of the protein in the sample based on the comparison in (c).   
     
     
         58 . The method of  claim 57 , wherein the sample is combined with the compound of  claim 2 . 
     
     
         59 . The method of  claim 57 , wherein the sample is combined with the compound of  claim 7 . 
     
     
         60 . The method of  claim 57 , wherein the sample is combined with the multidye composition of  claim 8 . 
     
     
         61 . The method of  claim 57 , wherein the sample is combined with the multidye composition of  claim 11 . 
     
     
         62 . The method of  claim 57 , wherein the comparison in step (c) is to the amount of fluorescence from a standard curve derived from measurements of protein aggregates and protein monomers in selected proportions. 
     
     
         63 . The method of  claim 62 , wherein the protein for the standard curve is the same protein as the protein in the sample. 
     
     
         64 . The method of  claim 62 , wherein the protein for the standard curve comprises a protein that is different from the protein in the sample. 
     
     
         65 . The method of  claim 57 , wherein the sample is from a mammal. 
     
     
         66 . The method of  claim 65 , wherein the sample is a bodily fluid of the mammal. 
     
     
         67 . The method of  claim 65 , wherein the sample comprises a cell from the mammal. 
     
     
         68 . The method of  claim 67 , wherein the cell is intact. 
     
     
         69 . The method of  claim 68 , wherein the intact cell is combined with the compound or multidye composition and the fluorescence is measured histologically. 
     
     
         70 . The method of  claim 69 , wherein the fluorescence is measured by visual observation. 
     
     
         71 . The method of  claim 69 , wherein the fluorescence is measured by quantifying the amount of fluorescent light emitted from the cell. 
     
     
         72 . The method of  claim 69 , wherein the cell is fixed. 
     
     
         73 . The method of  claim 69 , wherein the cell is living. 
     
     
         74 . The method of  claim 73 , wherein the cell is treated with a protein known to aggregate in cells. 
     
     
         75 . The method of  claim 74 , wherein the protein is an amyloid beta peptide. 
     
     
         76 . The method of  claim 75 , wherein the amyloid beta peptide is amyloid beta peptide 1-42. 
     
     
         77 . The method of  claim 74 , wherein the cell is also treated with a compound to determine the effect of the compound on the aggregation of the protein in the cell. 
     
     
         78 . The method of  claim 67 , wherein the cell from the mammal is part of a tissue of the mammal that is homogenized to prepare the sample. 
     
     
         79 . The method of  claim 65 , wherein the mammal has a disorder characterized by altered protein aggregation. 
     
     
         80 . The method of  claim 79 , wherein the protein is immunoglobulin, a DNA polymerase or a fragment thereof, α-synuclein, synphilin-1, TCRα, P23H mutant of rhodopsin, ΔF508 mutant of CFTR, amyloid-β, prion protein, Tau, SOD1, Ig light chains, ataxin-1, ataxin-3, ataxin-7, calcium channel, atrophin-1, androgen receptor, p62/sequestosome1 (SQSTM1), Pael receptor, serum amyloid A, transthyretin, β2-microglobulin, apolipoprotein A-1, gelsolin, atrial natriuretic factor, lysozyme, insulin, fibrinogen, crystallin, surfactant protein C, lactoferrin, βig-h3, PAPB2, corneodesmosin, neuroserpin, cochlin, RET, myelin, protein 22/0, SCAD, prolactin, lactadherin, p53, procalcitonin, cytokeratin, GFAP, ATP7B, prolyl hydroxylase PHD3, presenilin, or huntingtin. 
     
     
         81 . The method of  claim 79 , wherein the disorder is Alzheimer's disease, Huntington's disease, Parkinson's disease, senile systemic amyloidosis, or a spongiform encephalopathy. 
     
     
         82 . The method of  claim 57 , wherein the protein aggregates comprise aggresomes, aggresome-like structures, inclusion bodies, Lewy bodies, Mallory bodies, neurofibriliary tangles, or a combination thereof. 
     
     
         83 . The method of  claim 57 , wherein the amount of fluorescence is measured at preselected time intervals to detect formation of protein aggregates, wherein increasing fluorescence over time indicates formation of protein aggregates. 
     
     
         84 . The method of  claim 83 , wherein the preselected time intervals are less than 2 minutes. 
     
     
         85 . The method of  claim 83 , wherein the preselected time intervals are less than 10 minutes. 
     
     
         86 . The method of  claim 83 , wherein the preselected time intervals are less than 1 hour. 
     
     
         87 . The method of  claim 83 , wherein the preselected time intervals are more than 1 hour. 
     
     
         88 . The method of  claim 57 , wherein the dye-sample mixture of (a) is incubated at at least two different temperatures to determine the temperature dependency of protein aggregation. 
     
     
         89 . The method of  claim 88 , wherein the at least two different temperatures are between about 4° C. to about 100° C., inclusive. 
     
     
         90 . The method of  claim 57 , further comprising adding a test compound to a portion of the dye-sample mixture of (a) and comparing the fluorescence of the portion with the test compound to the fluorescence of the portion without the test compound to determine whether the test compound decreases aggregation of the protein, wherein decreased fluorescence in the portion with the test compound indicates that the test compound decreases aggregation of the protein. 
     
     
         91 . The method of  claim 90 , wherein the fluorescence of the portion with the test compound is compared to the fluorescence of the test compound at preselected time intervals. 
     
     
         92 . The method of  claim 91 , wherein the preselected time intervals are less than 2 minutes. 
     
     
         93 . The method of  claim 91 , wherein the preselected time intervals are less than 10 minutes. 
     
     
         94 . The method of  claim 91 , wherein the preselected time intervals are less than 1 hour. 
     
     
         95 . The method of  claim 91 , wherein the preselected time intervals are more than 1 hour. 
     
     
         96 . The method of  claim 90 , wherein the test compound comprises a kosmotrope, a chaotrope, an amino acid, a peptide, a reducing agent, a carbohydrate, a detergent, a surfactant, a zwitterion, a polyhydric alcohol, or a combination thereof. 
     
     
         97 . The method of  claim 90 , wherein the test compound is a storage buffer for the protein. 
     
     
         98 . The method of  claim 57 , wherein
 (A) the protein is a substrate for a chaperone;   (B) the dye-sample mixture of step (a) is subjected to a stress for a time and under conditions sufficient to induce aggregation of the protein; and   (C) the amount of fluorescence determined in (b) is compared to the amount of fluorescence from the protein with the compound or multidye composition subjected to the same stress without the sample,   wherein a decrease in fluorescence of the stressed dye-sample mixture with the sample when compared to the fluorescence from the protein with the compound or multidye composition but without the sample indicates that the sample comprises the chaperone.   
     
     
         99 . The method of  claim 98 , wherein the chaperone is a heat-shock protein. 
     
     
         100 . The method of  claim 99 , wherein the heat-shock protein is HSP33, HSP60, HSP70, HSP90, HSP100, or a combination thereof. 
     
     
         101 . The method of  claim 98 , wherein said chaperone is GRP94, GRP170, calnexin, calreticulin, HSP 40, HSP47 and ERp29, GroEL, GroES, HSP60, Cpn10, DnaK, DnaJ, Hsp70, Hsp71, Hsp72, Grp78 (BiP), PDI, Erp72, Hsx70, Hdj1, Hdj2, Mortalin, Hsc70, Hsp70-A1, fHtpG, C62.5, Hsp90α, Hsp90β, Grp94, ClpB, ClpA, ClpX, Hsp100, Hsp104, Hsp110, TRiC, α-crystallin, HspB1, Hsp 25, Hsp27, clusterin, GrpE, Trigger Factor, Survival of Motor Neuron (SMN1, SMN2), or a combination thereof. 
     
     
         102 . The method of  claim 98 , wherein the protein chaperone substrate is ß-lactoglobulin, citrate synthase, lysozyme, immunoglobulin, CRYBB2, HSPB8, CRYAA, TGFB1I1, HNRPD, CRYAB, or a combination thereof. 
     
     
         103 . The method of  claim 98 , wherein the stress is an elevated temperature. 
     
     
         104 . The method of  claim 103 , wherein the elevated temperature is from about 37° C. to about 95° C. 
     
     
         105 . The method of  claim 98 , wherein said stress is a chaotropic agent. 
     
     
         106 . The method of  claim 105 , wherein the chaotropic agent comprises guanidine-HCl, urea, or both. 
     
     
         107 . The method of  claim 57 , wherein
 (A) the protein is a substrate for an anti-chaperone; and   (B) the amount of fluorescence determined in (b) is compared to the amount of fluorescence from the protein with the compound or multidye composition without the sample,   wherein an increase in fluorescence of the dye-sample mixture when compared to the fluorescence from the protein with the compound or multidye composition but without the sample indicates that the sample comprises the anti-chaperone.   
     
     
         108 . The method of  claim 57 , wherein
 (A) the protein is a substrate for a member of the thioredoxin superfamily;   (B) a reducing agent is included in the dye-sample mixture of (a); and   (C) the mixture of step (a) is incubated for a period of time sufficient to reduce disulfide bonds in the protein,   wherein an increase in fluorescence of the dye-sample mixture when compared to the fluorescence from the protein with the compound or multidye composition without the sample indicates that the sample comprises the member of the thioredoxin superfamily.   
     
     
         109 . The method of  claim 108 , wherein the member of the thioredoxin superfamily comprises a protein disulfide isomerase, a thioredoxin, a glutaredoxin, or a combination thereof. 
     
     
         110 . The method of  claim 108 , wherein the member of the thioredoxin superfamily comprises a protein disulfide isomerase. 
     
     
         111 . The method of  claim 110 , wherein the protein disulfide isomerase comprises PDI, ERp57, PDIp, ERp72, P5, PDIr, ERp28/29, ERp44, ERjd5/JPDI, ERp18, or a combination thereof. 
     
     
         112 . The method of  claim 108 , wherein the substrate comprises insulin ribonuclease, choriogonadotropin, coagulation factor, glucocorticoid receptor, HIV gp120, or a combination thereof. 
     
     
         113 . The method of  claim 108 , wherein the reducing agent comprises dithiothreitol (DTT), Tris(2-carboxyethyl)phosphine hydrochloride (TCEP HCl), dithioerythritol (DTE), or a combination thereof. 
     
     
         114 . The method of  claim 108 , wherein the incubation is terminated by adding hydrogen peroxide to the combination prior to measuring the amount of fluorescence. 
     
     
         115 . A method for separating aggregates of a protein from monomeric forms of the protein in a sample, the method comprising
 (a) combining the sample to the solid support of  claim 39  under conditions where aggregates of the protein preferentially bind to the compound; and   (b) separating sample protein bound to the solid support from unbound protein,   wherein protein bound to the solid support are substantially aggregates and unbound protein is substantially monomers.