Selective capture and enrichment of proteins expressed on the cell surface

• US 20060154332A1
• Filed: 11/19/2004
• Published: 07/13/2006
• Est. Priority Date: 11/21/2003
• Status: Active Grant

• Current Assignee: Applied Biosystems LLC (Thermo Fisher Scientific Incorporated)
• Original Assignee: Applera Corporation (Thermo Fisher Scientific Incorporated)
• Inventors: Domon, Bruno, McCaffery, Ian, Ryan, Terence
• Granted Patent: US 7,348,416 B2
• Time in Patent Office: Days
• US Class Current: 435/69.100
• CPC Class Codes: C07K 1/14 Extraction; Separation; Purification
• CPC Class Codes: C07K 14/705 Receptors; Cell surface antigens; Cell surface determinants tumour specific antigens C07K14/4748

Abstract

The present invention provides a method for enriching cell surface proteins by oxidizing glycosylated protein with periodate. The present invention further provides that the cell surface proteins are obtained from intact cells either from cell lines or from tissues. The present invention provides a method not only capable of obtaining increased quantities of the cell surface proteins, but also capable of obtaining a wide ranges of proteins that are not detectable by other means.

Claims

1. A method for obtaining cell surface proteins comprising the steps of:

a) Mixing intact cell sample with a periodate forming a mixture,

b) Reacting the mixture with a binding reagent forming a binding complex, and

c) Isolating the proteins from the binding complex, wherein the isolated proteins are cell surface proteins.

2. The method of claim 1, wherein the intact cells are either cell lines or cells isolated from tissues.

3. The method of claim 1, wherein periodate is sodium periodate.

4. The method of claim 1, wherein the binding reagent is selected from a group consisting of hydrazide and amine.

5. The method of claim 1, wherein the binding reagent is on a resin or a solid support.

6. The method of claim 1, wherein the binding complex is further washed under a stringent condition to keep glycosylated cell surface proteins.

7. The method of claim 1, wherein the cell surface proteins are 50 fold enriched relative to total cellular proteins.

8. The method of claim 1, wherein the cell surface proteins are 100 fold enriched relative to total cellular proteins.

9. A binding complex comprising cell surface proteins of intact cells oxidized by periodate and covalently bond to a binding reagent that reacts to carbonyl groups of the cell surface proteins.

10. The complex of claim 9, wherein said binding reagent is selected from a group consisting of hydrazide and amine.

11. The complex of claim 10, wherein the binding reagent is hydrazide.

12. The complex of claim 9, wherein said periodate is selected from a group consisting of sodium periodate.

13. A method for obtaining cell surface proteins comprising the steps of:

a) Obtaining a homogenous cell population from a biological sample, which are intact cells,

b) Mixing intact cell sample with a periodate forming a mixture,

c) Reacting the mixture with a binding reagent forming binding complex, and

d) Isolating the proteins from the binding complex, wherein the isolated proteins are cell surface proteins.