Liquid tissue preparation from histopathologically processed biological samples, tissues and cells
The current invention provides a method for directly converting histopathologically processed biological samples, tissues, and cells into a multi-use biomolecule lysate. This method allows for simultaneous extraction, isolation, solublization, and storage of all biomolecules contained within the histopathologically processed biological sample, thereby forming a representative library of said sample. This multi-use biomolecule lysate is dilutable, soluble, capable of being fractionated, and used in any number of subsequent experiments.
1. A method of preparing a biomolecule lysate, comprising the steps of:
(a) heating a composition comprising a formalin fixed biological sample and a reaction buffer at a temperature from 80° C. to 100° C. for a period of time from 10 minutes to 4 hours to reverse or release protein cross-linking in said biological sample, and
(b) treating the resulting composition with an effective amount of a proteolytic enzyme selected from the group consisting of trypsin, chymotrypsin, and endoproteinase Lys-C for a period of time from 30 minutes to 24 hours at a temperature from 37° C. to 65° C. to disrupt the tissue and cellular structure of said biological sample and to liquefy said sample, thereby producing a liquid, soluble, dilutable biomolecule lysate that is suitable for protein analysis and wherein the protein content of said lysate is representative of the total protein content of said biological sample.
2. The method according to claim 1 , wherein said biological sample comprises a homogeneous population of tissues or cells.
3. The method according to claim 1 , further comprising, prior to step (a), the step of removing any paraffin present in said biological sample by one or more methods selected from the group consisting of: adding an organic solvent; heating; heating and adding a buffer comprising Tris; and heating and adding an organic solvent.
4. The method according to claim 1 , further comprising, prior to step (b), the step of mechanically disrupting said biological sample by at least one technique selected from the group consisting of: manual homogenization; vortexing; and physical mixing.
5. The method according to claim 1 , wherein said reaction buffer comprises a detergent.
6. The method according to claim 1 , wherein step (b) is carried out in the presence of a detergent.
7. The method according to claim 1 , wherein said reaction buffer comprises Tris and has a pH in the range of 1.0 to 9.0.
8. The method of claim 1 , further comprising assaying said biomolecule lysate using mass spectrometry.
9. The method according to claim 1 , wherein said reaction buffer comprises Tris and has a pH in the range of 6.0 to 9.0.
10. A method of preparing a biomolecule lysate, comprising the steps of:
(a) heating a composition comprising a formalin fixed biological sample and a reaction buffer at a temperature from 80° C. to 100° C. for a period of time from 10 minutes to 4 hours to reverse or release protein cross-linking in said biological sample,
(b) treating the resulting composition with an effective amount of a proteolytic enzyme selected from the group consisting of trypsin, chymotrypsin, and endoproteinase Lys-C for a period of time from 30 minutes to 24 hours at a temperature between 37° C. to 65° C. to disrupt the tissue and cellular structure of said biological sample and to liquefy said sample, thereby producing a liquid, soluble, dilutable biomolecule lysate that is suitable for protein analysis and wherein the protein content of said lysate is representative of the total protein content of said biological sample, and
fractionating said biomolecule lysate into distinct and separate biomolecule fractions.
11. The method according to claim 10 , wherein each biomolecule fraction contains distinct and separate biomolecules suitable for use in biochemical assays.
12. The method according to claim 1 , wherein said biological sample is selected from a group consisting of formalin-fixed tissue/cells, formalin-fixed/paraffin embedded (FFPE) tissue/cells, FFPE tissue blocks and cells from those blocks, and tissue culture cells that have been formalin fixed and or paraffin embedded.
13. The method of claim 10 , wherein said fractionating is carried out using a method selected from the group consisting of step spin column fractionation, immunoprecipitation, gradient centrifugation, HPLC and drip column fractionation.
14. The method according to claim 5 , wherein said detergent is selected from the group consisting of NONIDET P40, SDS, TWEEN 20, TRITON X, and sodium deoxycholate.
15. The method according to claim 6 , wherein said detergent is selected from the group consisting of NONIDET P40, SDS, TWEEN 20, TRITON X, and sodium deoxycholate.