Protein separation via multidimensional electrophoresis
First Claim
1. A method for separating a plurality of proteins in an initial sample, comprising(a) performing a plurality of capillary electrophoretic methods in series, each method comprising electrophoresing a sample obtained from the preceding method in the series, except the first method of the series in which the sample is the initial sample, and wherein at least one of the plurality of methods is capillary isoelectric focusing electrophoresis (CIEF) or capillary zone electrophoresis (CZE) and the at least one method is conducted under conditions such that electroosmotic flow is less than or equal to 0.5×
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6 cm2/V-s; and
(b) detecting resolved proteins from the final electrophoretic method.
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Abstract
The present method provides methods and apparatus for separating proteins using a series of electrophoretic methods that utilize controlled fractionation and labeling techniques to resolve mixtures of proteins. The samples for each electrophoretic method other than the initial method, contain only a subset of proteins resolved in the preceding method. The methods can be used in a variety of different applications including, creating proteomic databases, comparative expression studies, diagnostics, structure activity relationships and metabolic engineering investigations.
152 Citations
55 Claims
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1. A method for separating a plurality of proteins in an initial sample, comprising
(a) performing a plurality of capillary electrophoretic methods in series, each method comprising electrophoresing a sample obtained from the preceding method in the series, except the first method of the series in which the sample is the initial sample, and wherein at least one of the plurality of methods is capillary isoelectric focusing electrophoresis (CIEF) or capillary zone electrophoresis (CZE) and the at least one method is conducted under conditions such that electroosmotic flow is less than or equal to 0.5× - 10−
6 cm2/V-s; and
(b) detecting resolved proteins from the final electrophoretic method. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28)
the performing further comprises repeating the electrophoresing step multiple times for each electrophoretic method, each time with a different sample containing only a subset of the plurality of resolved proteins from the immediately preceding method in the series, whereby a plurality of resolved proteins for each of the different samples is obtained; - and
the detecting comprises detecting resolved proteins from each of the different samples from the final electrophoretic method.
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7. The method of claim 1, wherein the detecting comprises detecting resolved proteins contained in an electrophoretic medium as the electrophoretic medium passes a detector.
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8. The method of claim 1, wherein the detecting comprises detecting protein within a separation cavity utilized to perform the final electrophoretic method.
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9. The method of claim 1, further comprising quantifying resolved proteins from the final electrophoretic method.
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10. The method of claim 7, further comprising quantifying resolved protein from an electrical signal from the detector.
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11. The method of claim 1, further comprising analyzing resolved proteins from the final electrophoretic method by a technique other than electrophoresis.
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12. The method of claim 11, wherein the analyzing comprises conducting mass spectrometry with the resolved proteins.
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13. The method of claim 1, wherein at least one of the plurality of capillary electrophoretic methods utilizes a capillary adapted to reduce electroosmotic flow within the capillary.
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14. The method of claim 13, wherein the internal surface of the capillary is coated with an agent effective to reduce electroosmotic flow.
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15. The method of claim 14, wherein the agent is selected from the group consisting of polyacrylamide, polyethylene glycol and polyethylene oxide.
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16. The method of claim 13, wherein an induced counter current is generated to counteract electroosmotic flow within the capillary.
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17. The method of claim 13, wherein the capillary contains a porous plug positioned upstream from where sample is introduced.
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18. The method of claim 17, wherein the plug is composed of a polymeric material capable of forming a gel.
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19. The method of claim 18, wherein the polymeric material comprises polymerized acrylamide with diacrylamide crosslinkers or agarose.
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20. The method of claim 1, wherein one of the plurality of methods is capillary zone electrophoresis, and the electrophoresing is performed with an electrophoretic medium having a pH that reduces electroosmotic flow to a selected level.
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21. The method of claim 1, wherein the plurality of electrophoretic methods are capillary electrophoresis methods conducted with microcapillaries fabricated within a substrate.
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22. The method of claim 1, wherein one of the plurality of electrophoretic methods is capillary isoelectric focusing electrophoresis, and the electrophoresing is conducted with an electrophoretic medium lacking a polymeric agent.
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23. The method of claim 1, wherein one of the plurality of electrophoretic methods is capillary isoelectric focusing electrophoresis, and the electrophoresing is conducted with an electrophoretic medium containing a polymeric agent.
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24. The method of claim 1, wherein the electrophoresing step for each method comprises withdrawing the resolved proteins from an end of the capillary towards which proteins migrate during electrophoresing.
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25. The method of claim 1, wherein the electrophoresing step for each method comprises withdrawing the resolved proteins from different locations located between the two ends of the capillary.
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26. The method of claim 1, wherein the electrophoresing step for at least one of the plurality of capillary electrophoretic methods comprises eluting resolved proteins by electrophoretic mobility, electroosmotic flow or hydrodynamic pressure.
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27. The method of claim 1, further comprising analyzing the detected proteins to determine one or more chemical or physical characteristics of the detected proteins.
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28. The method of claim 27, wherein the one or more chemical or physical characteristics are selected from the group consisting of molecular weight, complete or partial amino acid sequence, isoelectric point, relative or absolute abundance and combinations of the foregoing.
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29. A method for separating a plurality of proteins in an initial sample, comprising
(a) performing a plurality of capillary electrophoretic methods in series, each method comprising electrophoresing a sample obtained from the preceding method in the series, except the first method of the series in which the sample is the initial sample, whereby a plurality of resolved proteins are obtained; -
(b) detecting resolved proteins from the final electrophoretic method;
(c) repeating steps (a) and (b) under the same conditions, wherein the initial sample for the first iteration of the method is from a first source and the initial sample for the second iteration of the method is from a second source; and
(d) comparing the relative abundance of one or more of the resolved proteins obtained from the first source and the second source as detected from the final electrophoretic method. - View Dependent Claims (30, 31)
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32. A method for separating a plurality of proteins, comprising
(a) performing a plurality of electrophoretic methods in series, wherein the method or methods preceding the final method comprise withdrawing and collecting multiple fractions containing proteins resolved during the electrophoretic method, and wherein each electrophoretic method is conducted with a sample from a fraction collected in the preceding electrophoretic method, except the first electrophoretic method which is conducted with a sample containing the plurality of proteins and the final electrophoretic method which is conducted with a sample from one or more fractions obtained in the penultimate electrophoretic method; -
(b) labeling the plurality of proteins or labeling protein contained in one or more collected fractions prior to conducting the last electrophoretic method; and
(c) detecting one or more labeled, resolved proteins obtained following the final electrophoretic method. - View Dependent Claims (33, 34, 35, 36)
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37. A method for separating a plurality of proteins, comprising
(a) performing one or more capillary electrophoretic methods, each of the one or more methods comprising (i) electrophoresing a sample containing multiple proteins within an electrophoretic medium contained within a capillary; - and
(ii) withdrawing and collecting multiple fractions containing proteins resolved during the electrophoresing step, and wherein each method is conducted with a sample from a fraction collected in the preceding electrophoretic method, except the first electrophoretic method which is conducted with a sample containing the plurality of proteins;
(b) labeling the plurality of proteins or labeling protein contained in one or more collected fractions prior to conducting the last electrophoretic method; and
(c) conducting a final capillary electrophoresis method comprising electrophoresing a sample obtained from one or more fractions obtained from the preceding method in a final capillary and detecting resolved protein within, or withdrawn from, the final capillary. - View Dependent Claims (38, 39, 40)
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41. A method for separating a plurality of proteins in an initial sample, comprising:
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(a) performing a plurality of electrophoretic methods in series, each method comprising electrophoresing within an electrophoretic medium a sample containing multiple proteins whereby fractions containing a subset of the multiple proteins are isolated physically, temporally or spatially, and wherein the sample electrophoresed is obtained from a fraction isolated during the immediately preceding method in the series, except the first method of the series in which the sample is the initial sample; and
(b) detecting isolated proteins from the final electrophoretic method;
(c) analyzing the isolated proteins to determine one or more chemical or physical characteristics of each of the isolated proteins; and
(d) storing the one or more chemical or physical characteristics into a database with information about the initial sample. - View Dependent Claims (42, 43)
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44. A method for separating a plurality of proteins in an initial sample, comprising:
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(a) performing a preliminary separation of the plurality of proteins by a non-electrophoretic technique to obtain multiple fractions, wherein each fraction contains a subset of the plurality of proteins and the preliminary separation separates at least some of the plurality of proteins according to differences in amino acid sequence;
(b) performing a plurality of electrophoretic methods in series, each method comprising electrophoresing a sample containing multiple proteins, whereby a plurality of resolved proteins are obtained, and wherein the sample electrophoresed contains only a subset of the plurality of resolved proteins from the immediately preceding method in the series, except the first electrophoretic method of the series in which the sample is from one of the multiple fractions; and
(c) detecting resolved proteins from the final electrophoretic method. - View Dependent Claims (45)
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46. A method for separating a plurality of proteins in an initial sample, comprising
(a) performing a plurality of capillary electrophoretic methods in series, wherein the method or methods preceding the final method comprises withdrawing and collecting multiple fractions containing proteins resolved during the electrophoretic method, each electrophoretic method is conducted with a sample from a fraction collected in the preceding electrophoretic method, except the first electrophoretic method which is conducted with a sample containing the plurality of proteins and the final electrophoretic method which is conducted with a sample from one or more fractions obtained in the penultimate electrophoretic method; - and
the plurality of capillary electrophoretic methods include at least one of the methods selected from the group consisting of capillary isoelectric focusing (CIEF), capillary zone electrophoresis (CZE) and capillary gel electrophoresis (CGE);
(b) labeling the proteins in the initial sample or labeling resolved protein(s) obtained from at least one of the capillary electrophoresis methods prior to conducting the last electrophoretic method, wherein if the plurality of methods includes a CIEF method, labeling is performed after the CIEF method;
if the plurality of methods includes a CZE method, labeling is performed before the CZE method; and
if the plurality of methods include a CGE method, labeling is performed before the CGE method; and
(c) detecting labeled proteins from the final electrophoretic method. - View Dependent Claims (47, 48, 49, 50, 51, 52)
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53. A method for separating a plurality of proteins in an initial sample, comprising:
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(a) performing a plurality of capillary electrophoretic methods in series, each method comprising electrophoresing a sample obtained from the preceding method in the series, except the first method of the series in which the sample is the initial sample, whereby one or more resolved proteins are obtained, and wherein at least one of the methods is a capillary isoelectric focusing method in which electrophoresing is performed in an electrophoretic medium and comprises eluting resolved proteins by electroelution, electroelution comprising (i) altering the salt concentration of the electrophoretic medium, and/or (ii) pH mobilization of resolved proteins; and
(b) detecting resolved protein from the final electrophoretic method. - View Dependent Claims (54, 55)
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Specification