Multiplexed measurement of membrane protein populations

• US 6,627,400 B1
• Filed: 10/27/2000
• Issued: 09/30/2003
• Est. Priority Date: 04/30/1999
• Status: Expired due to Term

• Current Assignee: Monogram Biosciences, Inc. (Laboratory Corporation Of America Holdings)
• Original Assignee: Aclara Biosciences, Inc (Laboratory Corporation Of America Holdings)
• Inventors: Singh, Sharat, Matray, Tracy
• Primary Examiner(s): Whisenant, Ethan
• Assistant Examiner(s): Tung, Joyce
• Application Number:
  US09/698,846
• Time in Patent Office: 1,068 Days
• Field of Search: 435/7, 435/7.2, 435/7.7, 435/7.72, 435/7.95
• US Class Current: 506/4
• CPC Class Codes: C07H 19/06 Pyrimidine radicals
• CPC Class Codes: C07H 19/10 with the saccharide radical esterified by phosphoric or polyphosphoric acids
• CPC Class Codes: C07H 21/00 Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• CPC Class Codes: C40B 20/08 Direct analysis of the library members per se by physical methods, e.g. spectroscopy
• CPC Class Codes: C40B 40/08 Libraries containing RNA or DNA which encodes proteins, e.g. gene libraries
• CPC Class Codes: C40B 50/16 involving encoding steps
• CPC Class Codes: C40B 70/00 Tags or labels specially adapted for combinatorial chemistry or libraries, e.g. fluorescent tags or bar codes

Abstract

Families of compositions are provided as labels, referred to as eTag reporters for attaching to polymeric compounds and assaying based on release of the eTag reporters from the polymeric compound and separation and detection. For oligonucleotides, the eTag reporters are synthesized at the end of the oligonucleotide by using phosphite or phosphate chemistry, whereby mass-modifying regions, charge-modifying regions and detectable regions are added sequentially to produce the eTag labeled reporters. By using small building blocks and varying their combination large numbers of different eTag reporters can be readily produced attached to a binding compound specific for the target compound of interest for identification. Protocols are used that release the eTag reporter when the target compound is present in the sample.

Claims

1. A method for determining populations of each of a plurality of surface membrane proteins in a cellular sample, the method comprising the steps of:

providing a binding compound for each of the plurality of surface membrane proteins, each binding compound having one or more eTag reporters attached thereto by a cleavable linkage, the one or more eTag reporters of each different binding compound having a different electrophoretic mobility so that eTag reporters of each different binding compound form distinct peaks upon electrophoretic separation;

combining with the cellular sample a binding compound for each of the plurality of surface membrane proteins such that in the presence of a surface membrane protein a complex is formed between each surface membrane protein and the binding compound specific therefor;

cleaving the cleavable linkage of each binding compound forming such complex so that eTag reporters are released; and

electrophoretically separating and identifying the released eTag reporters to determine the populations of the plurality of surface membrane proteins.

2. The method of claim 1 further including a step prior to said step of cleaving, the step comprising separating said complexes from unbound said binding compounds.

3. The method of claim 2 wherein said step of cleaving includes treating said cleavage linkage with an enzyme to release said eTag reporters.

4. The method of claim 2 wherein each of said eTag reporters has a fluorescent label or an electrochemical label.

5. The method of claim 1 wherein said binding compound is an antibody or fragment thereof.

6. The method of claim 5 wherein said released eTag reporters have a charge opposite that of said complexes and said binding compounds, wherein said cleavable linkage is cleaved by oxidation, and wherein said step of cleaving includes providing an active species for oxidizing said cleavable linkage.

7. The method of claim 5 wherein said cleavable linkage is cleaved by oxidation and wherein said step of cleaving includes providing an active species for oxidizing said cleavable linkage.

8. The method of claim 7 wherein said active species is selected from the group consisting of singlet oxygen, hydrogen peroxide, NADH, and hydroxyl radicals.

9. The method of claim 8 wherein said step of cleaving further includes providing for each of said plurality of said surface membrane proteins a second binding compound specific therefor, the second binding compound being conjugated with an active species producing moiety for generating said active species for oxidizing said cleavable linkage.

10. The method according to claim 6, 7, 8, or 9 wherein said active species is singlet oxygen, wherein said second binding compound is an antibody or fragment thereof, and wherein said cleavable linkage is an olefin, a thioether, a sulfoxide, or a selenium analog of the thioether or sulfoxide.

11. The method of claim 10 wherein said binding compound and said second binding compound are each antibodies.

12. A method for determining populations of each of a plurality of surface membrane proteins in a cellular sample, the method comprising the steps of:

providing a binding compound for each of a plurality of surface membrane proteins, each binding compound having one or more eTag reporters attached thereto by a cleavable linkage, the one or more eTag reporters of each different binding compound having a different charge/mass ratio so that eTag reporters of each different binding compound form distinct peaks upon electrophoretic separation;

providing a second binding compound for each of the plurality of surface membrane proteins, each second binding compound being conjugated with an active species producing moiety;

combining with the cellular sample a binding compound and a second binding compound for each of the plurality of surface membrane proteins such that in the presence of a surface membrane protein a complex is formed between the surface membrane protein and the binding compound and the second binding compound specific therefor, and such that the active species producing moiety of the second binding compound causes the generation of an active species and the cleavage of one or more cleavable linkages to release one or more eTag reporters; and

electrophoretically separating and identifying the released eTag reporters to determine the populations of surface membrane proteins in the cellular sample.

13. The method of claim 12 wherein said cleavable linkage is cleaved by oxidation and wherein said active species is singlet oxygen and wherein said active species producing moiety is a sensitizer.

14. The method of claim 13 wherein said cleavable linkage is an olefin, a thioether, a sulfoxide, or a selenium analog of the thioether or sulfoxide.

15. The method according to claim 12, 13, or 14 wherein said binding compound and said second binding compound are each antibodies or fragments thereof.