Protein Fractionation

• US 20020192759A1
• Filed: 05/22/2002
• Published: 12/19/2002
• Est. Priority Date: 05/22/2001
• Status: Active Grant

• Current Assignee: University of Vermont & State Agricultural College
• Original Assignee: University of Vermont & State Agricultural College
• Inventors: Maughan, David W.
• Granted Patent: US 7,172,739 B2
• Time in Patent Office: Days
• US Class Current: 435/69.1
• CPC Class Codes: C07K 1/20 Partition-, reverse-phase or hydrophobic interaction chromatography
• CPC Class Codes: C07K 1/36 by a combination of two or more processes of different types

Abstract

A method and apparatus for physically separating polypeptide constituents of a sample fluid by (a) providing a sample fluid comprising a mixture of polypeptides of differing physical and/or chemical properties; (b) contacting the fluid with at least two individual polymer matrix units, each polymer matrix unit preferentially accepting a different set of polypeptides on the basis of one or more physical and/or chemical properties; and (c) extracting the set of polypeptides from each polymer matrix unit.

Claims

1. A method for fractionating a sample of biological cellular material to separate a fraction of cellular components from the cellular material, the method comprising:

(a) providing a perforated perfusion chamber;

(b) placing a tissue sample in the chamber;

(c) exposing the tissue sample in the chamber to a fractionating fluid by;

(i) flowing a fractionating solution into the chamber at a rate calculated to permit fractionation of the sample;

or

(ii) flowing a fractionating solution into the chamber and maintaining the solution in the chamber for a time sufficient to permit fractionation of the sample;

(d) concurrently with (c)(i) or following (c)(ii), flowing fractionating fluid out of the chamber to form a bubble or droplet of fluid (or an array of bubbles or droplets) under oil, the fluid comprising a fraction of the cellular components of the cell.

2. The method of claim 1 wherein the sample of biological cellular material has a mass which is less than 1 mg.

3. The method of claim 1 wherein the sample of biological cellular material has a mass which is less than 0.1 mg.

4. The method of claim 1 further comprising enriching the polypeptide content of the sample fluid by removing water therefrom.

5. The method of claim 4 wherein the water is removed using a polymeric matrix unit which absorbs water with out absorbing polypeptides.

6. The method of claim 1 wherein steps 1(c) and 1(d) are repeated using fractionation solutions which extract different cellular components.

7. The method of claim 1 wherein one or more of the steps is automated.

8. The method of claim 1 wherein one or more of the steps is performed robotically.

9. The method of claim 1 wherein steps 1(c) and 1(d) are repeated using two or more solutions selected from the group consisting of:

(a) a solution which solubilizes plasma membrane while leaving membranes of intracellular organelles intact, permitting cytosolic polypeptides to diffuse out of cells of the sample and to be separated from non-cytosolic cellular material;

(b) a solution which solubilizes membranes of organelles, permitting organellar polypeptides to diffuse out of cells of the sample and to be separated from non-organellar cellular material; and

(c) a solution which solubilizes cytomatrix polypeptides permitting cytomatrix polypeptides to be separated from separated from non-cytomatrix cellular material.

10. The method of claim 9 wherein the solution of 9(a) comprises a detergent component.

11. The method of claim 10 wherein the detergent component comprises saponin.

12. The method of claim 9 wherein the solution of 9(b) comprises a detergent.

13. The method of claim 12 wherein the detergent component comprises triton X100.

14. The method of claim 9 wherein the solution of 9(c) comprises urea.

15. A method of physically separating polypeptides from an input composition comprising a mixture of polypeptides into two or more output compositions defined by physical and/or chemical properties, the method comprising:

(a) fractionating a sample of biological cellular material according to the method of claim 1 to provide a bubble of sample fluid comprising a mixture of polypeptides;

(b) providing two or more polymer matrix units, each of which preferentially excludes polypeptides on the basis of one or more physical or chemical properties;

(c) sequentially introducing each polymer matrix unit into the sample fluid;

(d) contacting a substrate with a polymer matrix unit, or vice versa, to deposit one or more sample aliquots for chemical analysis.

16. The method of claim 15 wherein one or more of the steps is automated.

17. The method of claim 15 wherein one or more of the steps is performed robotically.

19. The device of claim 18 wherein the perfusion chamber has a volume which is less than 10 μ

l.

20. The device of claim 18 wherein the perfusion chamber has a volume which is less than 1 μ

l.

21. The device of claim 18 wherein the perfusion chamber is in selective fluid communication with:

(a) a set of reservoirs, each comprising a fractionation solution;

(b) a set of one or more reservoirs, each comprising a wash solution.

22. The device of claim 21 wherein the solutions are selected from the group consisting of:

(a) a solution which solubilizes plasma membrane while leaving membranes of intracellular organelles intact permitting cytosolic polypeptides to diffuse out of cells of the sample and to be separated from non-cytosolic cellular material;

(b) a solution which solubilizes membranes of organelles permitting organellar polypeptides to diffuse out of cells of the sample and to be separated from non-organellar cellular material; and

(c) a solution which solubilizes cytomatrix polypeptides permitting cytomatrix polypeptides to be separated from separated from non-cytomatrix cellular material.

23. The device of claim 22 wherein the solution of 22(a) comprises a detergent component.

24. The device of claim 23 wherein the detergent component comprises saponin.

25. The device of claim 22 wherein the solution of 22(b) comprises a detergent.

26. The device of claim 25 wherein the detergent component comprises triton X100.

27. The device of claim 22 wherein the solution of 22(c) comprises urea.

28. A method for diagnosing a condition in a subject comprising:

obtaining a biological sample of tissue or cells from a subject,

fractionating the sample with the method of claim 1, and

determining the amount of one or more specific cellular components in the sample.

29. The method of claim 28, further comprising:

comparing the determination of the amount of specific cellular components in the sample and the amount of specific cellular components in a control group of cells as a diagnosis for a condition in the subject.

30. A method for determining onset, progression, or regression, of a disease in a subject, comprising:

obtaining a first biological sample of tissue or cells from a subject,

fractionating the sample with the method of claim 1,

determining the amount of one or more specific cellular components in the sample,

obtaining at a later time a second biological sample of tissue or cells from the subject,

determining the amount of one or more of the specific cellular components in the sample,

and comparing the determination of the one or more cellular components in the first sample and the second sample as a determination of the onset, progression, or regression of the disease.

31. A method for selecting a course of treatment of a subject having or suspected of having a disease, comprising:

obtaining from the subject a biological sample,

fractionating the sample with the method of claim 1,

determining at least one cellular component in the sample that is associated with the disease, and

selecting a course of treatment appropriate to the disease of the subject.

32. A method for evaluating the effect of candidate pharmacological compounds on a disease cell phenotype comprising:

obtaining a sample of cultured tissue or cells,

fractionating the sample with the method of claim 1,

determining the amount of one or more specific cellular components in the sample,

contacting the cultured tissue or cells with a candidate pharmacological agent,

obtaining a second sample of the cultured tissue or cells,

fractionating the second sample with the method of claim 1,

determining a second amount of one or more specific cellular components in the sample, and comparing the first and second amounts of one or more specific cellular components of the tissue or cells, wherein a change in the second amount of one or more specific cellular components, relative to the first amount of one or more specific cellular components, indicates the candidate pharmacological compound alters the amount of one or more specific cellular components indicating the onset of, progression of, or regression of a disease cell phenotype.

18. A perfusion chamber device for fractionating a sample of biological cellular material to separate a fraction of cellular components from the cellular material, the device comprising:

(a) a perfusion chamber for holding the sample, the perfusion chamber comprising one or more inlet ports for permitting fluid to enter the chamber and one or more outlet ports for permitting fluid to exit the chamber;

(b) a source of fractionating fluid and/or washing fluid in fluid communication with an inlet port;

(c) a means for forcing fluid from (b) into the perfusion chamber and out through the outlet port(s).