Difference detection methods using matched multiple dyes
First Claim
1. A method of comparing protein compositions between at least two different cell samples comprising:
- (a) preparing an extract of proteins from each of said at least two cell samples;
(b) providing a set of matched luminescent dyes chosen from dyes capable of covalently binding to proteins within said extract of proteins, wherein each dye within said set (1) has a net charge which will maintain the overall net charge of the proteins upon such covalent binding and has ionic and pH characteristics whereby relative migration of a protein labeled with any one of said dyes is the same as relative migration of said protein labeled with another dye in said set, (2) emits luminescent light at a wavelength that is sufficiently different from the emitted luminescent light of remaining dyes in said set to provide a detectably different light signal;
(c) reacting each extract of proteins of step (a) with a different dye from said set of step (b) to provide dye-labeled proteins;
(d) mixing each of said dye labeled proteins to form a single mixture of different dye-labeled proteins;
(e) separating the dye-labeled proteins of interest within said mixture; and
(f) detecting the difference in luminescent intensity between the different dye-labeled proteins of interest by luminescent detection.
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Abstract
A process and a kit are provided for detecting differences in two or more samples of protein, including proteins bearing post-translational modifications and peptides. Proteins are prepared, for example, from each of a different group of cell samples or body fluid samples to be compared. Each protein extract is labeled with a different one of a luminescent dye from a matched set of dyes. The matched dyes have generally the same ionic and pH characteristics but emit light at different wavelengths to exhibit a different color upon luminescence detection. The labeled protein extracts are mixed together and separated together by electrophoresis or a chromatographic method. The separation is observed to detect proteins unique to one sample or present in a greater ratio in one sample than in the other. Those unique or excess proteins will fluoresce the color of one of the dyes used. Proteins common to each sample migrate together and fluoresce the same.
37 Citations
39 Claims
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1. A method of comparing protein compositions between at least two different cell samples comprising:
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(a) preparing an extract of proteins from each of said at least two cell samples;
(b) providing a set of matched luminescent dyes chosen from dyes capable of covalently binding to proteins within said extract of proteins, wherein each dye within said set (1) has a net charge which will maintain the overall net charge of the proteins upon such covalent binding and has ionic and pH characteristics whereby relative migration of a protein labeled with any one of said dyes is the same as relative migration of said protein labeled with another dye in said set, (2) emits luminescent light at a wavelength that is sufficiently different from the emitted luminescent light of remaining dyes in said set to provide a detectably different light signal;
(c) reacting each extract of proteins of step (a) with a different dye from said set of step (b) to provide dye-labeled proteins;
(d) mixing each of said dye labeled proteins to form a single mixture of different dye-labeled proteins;
(e) separating the dye-labeled proteins of interest within said mixture; and
(f) detecting the difference in luminescent intensity between the different dye-labeled proteins of interest by luminescent detection. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18)
wherein the dotted lines each represent carbon atoms necessary for the formation of one to three fused rings having five to six atoms in each ring, X and Y are selected from the group consisting of S, O and CH3—
C—
CH3, m is an integer from 1 to 3, one of R1 and R2 is a reactive group and the other is an alkyl.
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4. The method of claim 1 wherein each dye has at least one reactive group selected from the group consisting of isothiocyanate, isocyanate, N-hydroxysuccinimidyl ester, imido ester, glyoxal, carboxylic acid, haloacetamide, maleimide, alkyl halide, acid halide, azide, hydrazide, hydrazine, ketone and amino.
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5. The method of claim 1 wherein said set of matched luminescent dyes are neutral dyes bound to the primary amino of a lysine residue in the protein by a positively charged linker group.
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6. The method of claim 1 wherein said set of matched luminescent dyes are neutral dyes bound to a sulfhydryl group in the protein.
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7. The method of claim 1 wherein said set of matched luminescent dyes are derivatives of dipyrromethene boron difluoride dyes.
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8. The method of claim 1 wherein separating the dye-labeled proteins is by an electrophoretic method.
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9. The method of claim 8 wherein the electrophoretic method comprises one dimensional gel electrophoresis, two dimensional gel electrophoresis, capillary zone electrophoresis, capillary gel electrophoresis, capillary isoelectric focusing, isotacophoresis, or micellar electrokinetic chromatography.
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10. The method of claim 1 wherein separating the dye-labeled proteins is by a chromatographic method.
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11. The method of claim 10 wherein the chromatographic method comprises affinity chromatography, size exclusion chromatography, reverse phase chromatography, hydrophobic interaction chromatography or ion exchange chromatography.
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12. The method of claim 1 further comprising quenching the reaction between the proteins and the dyes prior to mixing said dye labeled proteins.
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13. The method of claim 1 wherein the step of detecting differences in emitted color is by fluorescent microscopy.
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14. The method of claim 1 wherein the step of detecting differences in emitted color is by electronic imaging.
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15. The method of claim 1 wherein the proteins have binding sites for covalent binding to said dyes selected from the group consisting of lysine, carboxylic acid and sulfhydryl groups.
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16. The method of claim 1 wherein the proteins include glycoproteins having terminal sugar groups and preparing the extract of proteins further comprises the step of oxidizing terminal sugar groups to form aldehyde groups;
- and each dye of said set has a reactive group capable of forming a covalent bond with the aldehyde group.
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17. The method of claim 1 wherein the proteins include phosphoproteins.
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18. The method of claim 1 wherein preparing the extract of proteins further comprises digesting at least a portion of the proteins with enzyme for generating peptides.
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19. A method of comparing proteins of interest between at least two different samples comprising:
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(a) preparing a mixture of proteins from each of said at least two samples;
(b) providing a set of matched luminescent dyes chosen from dyes capable of covalently binding to said proteins within said mixture of proteins, wherein each dye within said set (1) has ionic and pH characteristics whereby relative migration of a protein labeled with any one of said dyes is the same as relative migration of said protein labeled with another dye in said set, (2) emits luminescent light at a wavelength that is sufficiently different from the emitted luminescent light of remaining dyes in said set to provide a detectably different light signal;
(c) reacting each said mixture of proteins of step (a) with a different dye from said set of step (b) to provide dye-labeled proteins;
(d) mixing each of said dye labeled proteins to form a combined single mixture of different dye-labeled proteins;
(e) separating the different dye-labeled proteins of interest within said combined mixture; and
(f) detecting the difference in luminescent intensity between the different dye-labeled proteins of interest by luminescent detection. - View Dependent Claims (20, 21, 22, 23, 24, 25, 26, 27, 28, 29)
wherein the dotted lines each represent carbon atoms necessary for the formation of one to three fused rings having five to six atoms in each ring, X and Y are each selected from the group consisting of O, S and CH3—
C—
CH3, m is an integer from 1 to 3, one of R1 and R2 is a reactive group and the other is an alkyl.
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21. The method of claim 19 wherein each dye has at least one reactive group selected from the group consisting of isothiocyanate, isocyanate, N-hydroxysuccinimidyl ester, imido ester, glyoxal, carboxylic acid, haloacetamide, maleimide, alkyl halide, acid halide, azide, hydrazide, hydrazine, ketone and amino.
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22. The method of claim 19 wherein the proteins have binding sites selected from the group consisting of lysine, carboxylic acid and sulfhydryl groups.
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23. The method of claim 19 wherein separating the dye-labeled proteins is by an electrophoretic method.
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24. The method of claim 23 wherein the electrophoretic method comprises one dimensional gel electrophoresis, two dimensional gel electrophoresis, capillary zone electrophoresis, capillary gel electrophoresis, capillary isoelectric focusing, isotacophoresis, or micellar electrokinetic chromatography.
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25. The method of claim 19 wherein separating the dye-labeled proteins is by a chromatographic method.
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26. The method of claim 25 wherein the chromatographic method comprises affinity chromatography, size exclusion chromatography, reverse phase chromatography, hydrophobic interaction chromatography or ion exchange chromatography.
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27. The method of claim 19 wherein the proteins are glycoproteins having terminal sugar groups and preparing the extract of proteins further comprises the step of oxidizing terminal sugar groups to form aldehyde groups;
- and each dye of said set has a reactive group capable of forming a covalent bond with the aldehyde group.
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28. The method of claim 19 wherein the proteins are phosphoproteins and the dye includes an imidazole group, said method further comprising adding carbodiimide to said mixture of proteins and dyes for the reaction of step (c).
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29. The method of claim 19 wherein preparing the extract of proteins further comprises digesting at least a portion of the proteins with enzyme for generating peptides.
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30. A method of comparing proteins of interest between at least two different samples comprising:
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(a) preparing a mixture of proteins from each of said at least two samples;
(b) providing a set of matched luminescent dyes chosen from a single class of dyes capable of covalently binding to proteins within said mixture of proteins, wherein each dye within said set (1) has ionic and pH characteristics whereby relative migration of a protein labeled with any one of said dyes is the same as relative migration of said protein labeled with another dye in said set, (2) emits luminescent light at a wavelength that is sufficiently different from the emitted luminescent light of remaining dyes in said set to provide a detectably different light signal;
(c) reacting each mixture of proteins of step (a) with a different dye from said set of step (b) to provide dye-labeled proteins;
(d) mixing each of said dye labeled proteins to form a combined single mixture of different dye-labeled proteins;
(e) separating different dye-labeled proteins of interest within said combined mixture; and
(f) detecting the difference in luminescent intensity between the different dye-labeled proteins of interest by luminescent detection. - View Dependent Claims (31, 32, 33, 34, 35, 36, 37, 38)
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39. A method of comparing protein compositions between at least two different cell samples comprising:
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(a) preparing an extract of proteins from each of said at least two cell samples;
(b) providing a set of matched luminescent dyes chosen from dyes capable of covalently binding to proteins within said extract of proteins, wherein each dye within said set (1) has a net charge which will maintain the overall net charge of the proteins upon such covalent binding and has ionic and pH characteristics whereby relative migration of a protein labeled with any one of said dyes is the same as relative migration of said protein labeled with another dye in said set, and, (2) emits luminescent light at a wavelength that is sufficiently different from the emitted luminescent light of remaining dyes in said set to provide a detectably different light signal;
(c) reacting each extract of proteins of step (a) with a different dye from said set of step (b) to provide dye-labeled proteins;
(d) mixing said dye labeled proteins to form a single mixture of different dye-labeled proteins;
(e) separating the dye-labeled proteins within said mixture by a chromatographic method; and
(f) detecting the difference in luminescent intensity between the different dye-labeled proteins of interest by luminescent detection.
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Specification