Methods and kits for isolation and analysis of a chromatin region
The present invention encompasses methods of identifying proteins and protein modifications of proteins specifically associated with a chromatin.
1. A method of identifying proteins including proteins comprising posttranslational modifications specifically associated with a target chromatin in a cell, the method comprising:
(a) providing:
i) a first cell sample comprising nucleic acid binding proteins and the target chromatin, wherein the target chromatin is tagged, and
ii) a second cell sample comprising nucleic acid binding proteins and the target chromatin, wherein the target chromatin is not tagged,
wherein the proteins in the first cell sample or the second cell sample are metabolically labeled,
(b) mixing the first cell sample and the second cell sample from (a) with an affinity handle to generate a cell mixture wherein the affinity handle binds protein such that the affinity handle specifically binds the tagged target chromatin and non-specifically binds nucleic acid binding proteins,
(c) isolating the affinity handle from the mixture in (b) wherein the affinity handle isolated consists of affinity handle bound to tagged target chromatin and affinity handle bound to nucleic acid binding proteins, wherein isolating the affinity handle isolates proteins associated with the tagged target chromatin,
(d) identifying isolated proteins from (c),
(e) determining whether each protein from (c) is metabolically labeled, unlabeled, or a mixture of labeled and unlabeled protein, wherein
i) if the first cell sample from (a) is not metabolically labeled and a protein associated with the target chromatin is not metabolically labeled, then the protein is specifically associated with the tagged chromatin in the cell, and
ii) if the first sample from (a) is metabolically labeled and a protein associated with the target chromatin is metabolically labeled, then the protein is specifically associated with the tagged chromatin in the cell.
2. The method of claim 1 , wherein the first cell sample is lysed before tagging the target chromatin.
3. The method of claim 1 , wherein the first cell sample is tagged before lysing the target chromatin.
4. The method of claim 3 , wherein the target chromatin is tagged by expressing protein A in a cell comprising the lexA nucleic acid binding site in the target chromatin.
5. The method of claim 1 , wherein the proteins in the first cell sample are metabolically labeled.
6. The method of claim 1 , wherein the proteins in the second cell sample are metabolically labeled.
7. The method of claim 1 , wherein the proteins in the first cell sample or the second cell sample are metabolically labeled with a heavy isotope.
8. The method of claim 1 , wherein the proteins in the first cell sample or the second cell sample are labeled by culturing cells in the presence of a labeled amino acid.
9. The method of claim 8 , wherein the labeled amino acid is [ 13 C6, 15 N4]-L-Arginine.
10. The method of claim 1 , wherein the proteins in the first cell sample and the second cell sample are crosslinked before lysis.
11. The method of claim 1 wherein the first cell sample and the second cell sample are mixed at a ratio of 1:100 to 100:1.
12. The method of claim 1 wherein the first cell sample and the second cell sample are mixed at a ratio of 1:1.
13. The method of claim 1 , wherein the chromatin in the lysate from step (b) is fragmented before isolating the tagged target nucleic acid sequence.
14. The method of claim 13 , wherein the chromatin in the lysate is fragmented to comprise nucleic acid sections comprising 500 to 1500 base pairs.
15. The method of claim 1 , wherein the tagged target nucleic acid sequence is isolated using affinity purification.
16. The method of claim 1 , wherein proteins associated with the target chromatin are identified using mass spectrometry.
17. The method of claim 1 , wherein mass spectrometry is used to determine if an identified protein associated with the target chromatin is labeled, unlabeled, or a combination of labeled and unlabeled protein.
18. The method of claim 1 , wherein if the first cell sample from (a) is not metabolically labeled and a protein associated with the target chromatin is metabolically labeled, or a combination of labeled and unlabeled protein, then the protein is not specifically associated with the tagged chromatin in the cell.
19. The method of claim 1 , wherein if the first cell sample from (a) is metabolically labeled and a protein associated with the target chromatin is not metabolically labeled, or a combination of labeled and unlabeled protein, then the protein is not specifically associated with the tagged chromatin in the cell.