Detecting RNAi using SELDI mass spectrometry

• US 20060063161A1
• Filed: 09/22/2004
• Published: 03/23/2006
• Est. Priority Date: 09/22/2004
• Status: Abandoned Application

• Current Assignee: Ciphergen Biosystems Incorporated
• Original Assignee: Ciphergen Biosystems Incorporated
• Inventors: Rich, William E., Reddy, Gurucharan
• Application Number:
  US10/948,028
• Publication Number:
  US 20060063161A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: G01N 33/6848 Methods of protein analysis involving mass spectrometry

Abstract

The present invention relates to the fields of protein expression and molecular biology. In particular, the present invention includes methods for monitoring the levels of polypeptide inhibition caused by inhibitory nucleic acids.

Claims

1. A method comprising:

a. expressing polypeptides in a first translation system, wherein the first translation system comprises at least one inhibitory nucleic acid directed toward at least one mRNA; and

b. measuring expression of at least one polypeptide in the translation system by affinity mass spectometry.

2. The method of claim 1 wherein the translation system is an in vitro translation system.

3. The method of claim 1 wherein the translation system is an in vivo translation system.

4. The method of claim 1 wherein the translation system is in contact with an affinity surface of an affinity mass spectrometry probe, whereby the affinity surface captures polypeptides expressed by the translation system in situ.

5. The method of claim 1 wherein the inhibitory nucleic acid is selected from an antisense nucleic acid, a ribozyme and an siRNA.

6. The method of claim 5 wherein the inhibitory nucleic acid is an siRNA comprising between 18 and 25 paired bases.

7. The method of claim 1 wherein the expression of at least one polypeptide is inhibited by the at least one inhibitory nucleic acid.

8. The method of claim 1 wherein the at least one inhibitory nucleic acid inhibits expression of a cell receptor selected from a nuclear receptor, a cytoplasmic receptor and a cell surface receptor.

9. The method of claim 8 wherein at least one of the polypeptides is a ligand for the cell receptor.

10. The method of claim 1 wherein the mRNA encodes a component of a signaling pathway.

11. The method of claim 9 wherein at least one of the polypeptides is a component of the pathway.

12. The method of claim 1 wherein the mRNA encodes a kinase or a phosphatase.

13. The method of claim 12 wherein at least one of the polypeptides is a biochemical substrate for the kinase or phosphatase.

14. The method of claim 1 wherein the mRNA encodes a protease.

15. The method of claim 14 wherein at least one of the polypeptides is a biochemical substrate for the protease.

16. The method of claim 1 wherein at least one polypeptide is a polypeptide that interacts with a polypeptide encoded by the mRNA.

17. The method of claim 1 wherein affinity mass spectrometry comprises capturing the at least one polypeptide on a mass spectrometry probe comprising a surface having a chromatographic capture reagent attached thereto.

18. The method of claim 1 wherein affinity mass spectrometry comprises capturing the at least one polypeptide on a mass spectrometry probe comprising a surface having a biospecific capture reagent attached thereto, wherein the biospecific capture reagent binds the polypeptide.

19. The method of claim 1 wherein affinity mass spectrometry further comprises SEND.

20. The method of claim 1 further comprising:

c. expressing second polypeptides in a second translation system, wherein the expression system does not comprise the at least one siRNA;

d. measuring expression of at least one of the second polypeptides in the expression system by affinity mass spectrometry; and

,

e. comparing expression of the polypeptides in the first and second expression systems.

21. The method of claim 20 comprising:

i. performing steps (a) and (b) on a plurality of first expression systems and measuring a plurality of first polypeptides;

ii. performing steps (c) and (d) on a plurality of second expression systems measuring a plurality of second polypeptides; and

iii. wherein comparing comprises using the measurements as a learning set to train a learning algorithm, thereby generating a classification model, wherein the classification model uses measurement of at least one polypeptide to classify an unknown sample as belonging to the first or second expression system.

22. A kit comprising:

a. A first expression system capable of expressing at least one inhibitory nucleic acid; and

b. At least one affinity mass spectrometry probe.

23. The kit of claim 22 further comprising instructions to use the kit to detect expression of a polypeptide whose expression is inhibited by the inhibitory nucleic acid.

24. The kit of claim 22 further comprising a second expression system that does not express the inhibitory nucleic acid.

25. The kit of claim 24 further comprising instructions to compare expression of a polypeptide in both the first and second expression systems.

26. A kit comprising:

a. at least one inhibitory nucleic acid capable of inhibiting expression of at least one expressed protein of a species; and

,

b. at least one affinity mass spectrometry probe capable of binding at least one of the expressed proteins.