Isolation of analytes
The disclosure features, inter alia, methods and compositions for detecting, isolating, and/or analyzing one or more analytes such as, but not limited to, biomolecules (e.g., proteins, glycoproteins, lipids (e.g., sphingolipids), and carbohydrates). Such methods and compositions are useful in, e.g., the discovery of novel proteins, or the amino acid sequence of novel proteins, or identifying biomarkers for diseases such as cancer, immunological disorders, or microbial infections.
1. A method for isolating an analyte from a mixture, the method comprising:
providing a mixture comprising an iminobiotin marker-conjugated analyte;
separating the iminobiotin marker-conjugated analyte from the mixture using a separation matrix;
labeling the iminobiotin marker-conjugated analyte in the separation matrix with a detectable biotin-binding molecule, wherein the labeling occurs under conditions sufficient for the biotin-binding molecule to bind to iminobiotin with high affinity;
isolating the iminobiotin marker-conjugated analyte from the separation matrix; and
removing the detectable biotin-binding molecule from the iminobiotin marker-conjugated analyte under conditions that decrease the binding affinity of the biotin-binding molecule for iminobiotin.
2. The method of claim 1 , wherein the mixture comprises more than one iminobiotin marker-conjugated analyte.
3. A method for isolating an analyte from a mixture, the method comprising:
conjugating an iminobiotin marker to an analyte in a mixture;
separating the iminobiotin marker-conjugated analyte from the mixture using a separation matrix;
labeling the iminobiotin marker-conjugated analyte in the separation matrix with a detectable biotin-binding molecule, wherein the labeling occurs under conditions sufficient for the biotin-binding molecule to bind to iminobiotin with high affinity;
isolating the iminobiotin marker-conjugated analyte from the separation matrix; and
removing the detectable biotin-binding molecule from the iminobiotin marker-conjugated analyte under conditions that decrease the binding affinity of the biotin-binding molecule for iminobiotin.
4. The method of claim 3 , wherein the iminobiotin marker is conjugated to more than one analyte.
5. The method of claim 4 , wherein more than one iminobiotin marker-conjugated analyte is isolated from the separation matrix.
6. The method of claim 4 , wherein the detectable biotin-binding molecule is removed from more than one iminobiotin marker-conjugated analyte.
7. The method of claim 3 , wherein the method comprises: isolating the iminobiotin marker-conjugated analyte from the separation matrix prior to removing the detectable biotin-binding molecule from the iminobiotin marker-conjugated analyte under conditions that decrease the binding affinity of the biotin-binding molecule for iminobiotin.
8. The method of any of claims 3 , wherein the method comprises: removing the detectable biotin-binding molecule from the iminobiotin marker-conjugated analyte under conditions that decrease the binding affinity of the biotin-binding molecule for iminobiotin prior to isolating the iminobiotin marker-conjugated analyte from the separation matrix.
9. The method of claim 3 , wherein the conditions sufficient for the biotin-binding molecule to bind to iminobiotin with high affinity are basic conditions.
10. The method of any of claims 3 , wherein the conditions that decrease the binding affinity of the biotin-binding molecule to iminobiotin are acidic conditions.
11. The method of any of claims 3 , wherein the separating comprises resolving the iminobiotin marker-labeled analytes in two dimensions.
12. The method of any of claims 3 , wherein the separation matrix is an acrylamide gel.
13. The method of claim 3 , further comprising concentrating the iminobiotin marker-conjugated analyte.
14. The method of claim 13 , wherein the concentrating occurs before separating the iminobiotin marker-conjugated analyte from the mixture using the separation matrix.
15. The method of claim 14 , wherein the concentrating comprises contacting the mixture with a biotin-binding column.
16. The method of claim 3 , wherein the detectable biotin-binding molecule is conjugated to a detectable label.
17. The method of claim 16 , wherein the detectable label is selected from the group consisting of an enzymatically active label, a fluorescent label, a luminescent label, and a colorimetric label.
18. The method of claim 3 , wherein the biotin-binding molecule is selected from the group consisting of neutravidin, captavidin, streptavidin, and avidin.
19. The method of claim 3 , wherein the iminobiotin marker-conjugated analyte is a polypeptide.
20. The method of claim 19 , wherein the polypeptide is a cell-surface polypeptide.
21. The method of claim 3 , further comprising, after separating the iminobiotin marker-conjugated analyte from the mixture using a separation matrix, fixing the iminobiotin marker-conjugated analyte in the separation matrix with a fixing agent.
22. The method of claim 21 , wherein the fixing agent is isopropanol.
23. The method of claim 3 , further comprising, after labeling the iminobiotin marker-conjugated analyte with a detectable biotin-binding molecule, detecting the iminobiotin marker-conjugated analyte.
24. The method of claim 3 , further comprising analyzing the isolated iminobiotin marker-conjugated analyte.
25. The method of claim 24 , wherein the analyzing comprises mass spectrometry.
26. The method of claim 24 , wherein the analyzing comprises detecting if the iminobiotin marker-conjugated analyte is modified.
27. The method of claim 3 , further comprising, after separating the iminobiotin marker-conjugated analyte from the mixture, determining the amount of iminobiotin marker-conjugated analyte.
28. The method of claim 3 , wherein the mixture is a cell lysate.
29. The method of claim 3 , wherein the iminobiotin marker is conjugated to the one or more analytes at the surface of a cell.
30. The method of claim 29 , further comprising after conjugating the iminobiotin marker to the one or analytes, preparing a mixture from the cell.