Methods for isolating proteins
The invention generally relates to methods for isolating proteins. In certain aspects, methods of the invention involve preparing a plurality of sample preparations, each preparation including one or more intact cells. A capture unit is introduced to a plurality of the preparations. The capture unit includes a member that transiently interacts with one or more proteins in the cells and a reactive functional group. The sample preparations are incubated, and a reaction is initiated at a different time in a plurality of the preparation. In this manner, a protein within the cell that specifically interacts with the member of the capture unit becomes bound to the capture unit via the reactive functional group to form protein/capture unit complexes. The cells are lysed and the protein/capture unit complexes are isolated.
1. A method for isolating a plurality of proteins, the method comprising:
preparing a plurality of sample preparations, each preparation comprising one or more intact cells;
introducing a capture unit to a plurality of the preparations, the capture unit comprising a member that transiently interacts with one or more proteins in the cells and a reactive functional group;
incubating the sample preparations;
initiating a reaction at a different time points in a plurality of the preparations such that a protein within the cell that specifically interacts with the member of the capture unit becomes bound to the capture unit via the reactive functional group to form protein/capture unit complexes;
lysing the cells according to the different time points at the initiating reaction step; and
isolating the protein/capture unit complexes.
2. The method according to claim 1 , wherein the member is capable of entering the cells through an entrance pathway of the cell.
3. The method according to claim 1 , wherein reactive functional moiety does not interfere with the specific interaction between the member and the proteins within the cell.
4. The method according to claim 3 , wherein the member is a nanoparticle.
5. The method according to claim 3 , wherein the member is a microorganism.
6. The method according to claim 5 , wherein the microorganism is a virus.
7. The method according to claim 1 , wherein the member is a protein.
8. The method according to claim 1 , wherein the reactive functional group comprises a photoreactive functional group.
9. The method according to claim 8 , wherein light is used to initiate the reaction in each preparation.
10. The method according to claim 1 , wherein the reactive functional group is a primary amine.
11. The method according to claim 10 , wherein a chemical is used to initiate the reaction in each preparation.
12. The method according to claim 11 , wherein the chemical is selected from the group consisting of formaldehyde, disuccinimidyl suberate (DSS), and Dithiobis[succinimidyl propionate] (DSP).
13. The method according to claim 1 , wherein the capture unit further comprises a purification handle.
14. The method according to claim 13 , wherein the capture units are separated from each sample preparation via the purification handle.
15. The method according to claim 1 , further comprising analyzing the separated proteins.
16. The method according to claim 15 , wherein the analyzing step uses a mass spectrometer.
17. The method according to claim 15 , wherein analyzing is by liquid chromatography —mass spectrometry (LC-MS).
18. The method according to claim 15 , wherein the capture unit further comprises a detectable label.
19. The method according to claim 18 , wherein the label is an optically detectable label.
20. The method according to claim 1 , wherein the reaction is initiated in a first sample preparation after 30 minutes of incubation, the reaction is initiated in a second sample preparation after 60 minutes of incubation, and the reaction is initiated in a third sample preparation after 120 minutes of incubation.
21. The method according to claim 1 , wherein the member also transiently interacts with proteins outside of the cell.
22. The method according to claim 21 , wherein the proteins are cell surface proteins.