Claims for Patent: 7,714,276

Summary for Patent: 7,714,276

Title:	Methods for direct biomolecule identification by matrix-assisted laser desorption ionization (MALDI) mass spectrometry
Abstract:	The present invention relates to the use of post source decay (PSD) or collision induced dissociation (CID) direct tissue (DT) MALDI-TOF or DT-MALDI-TOF-TOF mass spectrographic identification of biological molecules in a tissue or cellular sample without the need for further protein extraction. This method provides for studying cells or tissues by direct tissue MALDI (DT-MALDI), thereby substituting in situ protein release for further protein extraction. Mass/intensity data was processed with Mascot.COPYRGT. software interrogation of the NCBI database. These results are proof of principle that DT-MALDI, combined with bioinformatics, can directly identify proteins in cells and tissues from their mass spectra.
Inventor(s):	Pevsner; Paul (New York, NY), Naftolin; Frederick (Woodbridge, CT), Miller; Douglas C. (Delle Mead, NJ), Hillman; Dean (New Hyde Park, NY), Stall; Brian K. (Bedford, NH), Wishnie; Steven M. (Columbia, MD)
Assignee:	New York University (New York, NY)
Application Number:	11/541,380
Patent Claims:	1. A method for analyzing the biological molecule content of a tissue sample in situ, comprising: a. collecting a sample of tissue from a subject into a first solution capable of maintaining integrity of the biological molecule; b. treating the sample with a second solution comprising one or more enzymes, or chemicals, capable of dissociating the tissue sample or of digesting the dissociated tissue sample into smaller fragments; c. treating the preparation from step b) with a matrix assisted laser desorption ionization imaging (MALDI) matrix solution; and d. analyzing the preparation from step c) by direct tissue (DT)-matrix assisted laser desorption ionization imaging (MALDI)-time of flight (TOF) measurement or DT-MALDI-TOF-TOF measurement; e. creating a data file utilizing the information from step d); f. entering the data from the data file of step e) into an external database to create a signature map for the tissue from which the data was obtained; and g. comparing the results from step d) with a signature map for normal tissue, wherein said normal tissue corresponds to, or is of the same tissue type, as the tissue from which the sample was obtained. 2. The method of claim 1, wherein the method provides for both qualitative identification of the biological molecules in the sample, as well as, a quantitative measurement of the proteins biological molecules in the sample. 3. The method of claim 1, wherein the method provides for a level of detection of the biological molecules in the sample in an amount ranging from about 1 attamol to about 10 attamols. 4. The method of claim 1, wherein the collection device is a microcapillary pipette, a plastic or glass tube, or a slide for a cellular or tissue sample obtained from a microtime or an ultra cryo microtome. 5. The method of claim 1, wherein the first solution is a buffered solution or an alcohol. 6. The method of claim 5, wherein the buffered solution is selected from the group consisting of phosphate buffered saline (PBS), a phosphate buffer, a potassium buffer, a choline buffer and a glycine buffer. 7. The method of claim 5, wherein the alcohol is selected from the group consisting of methanol, ethanol, propanol, butanol, isopropyl alcohol and isobutanol. 8. The method of claim 1, wherein the one or more enzymes capable of dissociating the tissue and degrading the tissue into peptide fragments are selected from the group consisting of a collagenase, a lipase and a protease. 9. The method of claim 8, wherein the one or more enzymes are left in contact for a time and at a temperature sufficient to obtain dissociated tissue and peptide fragments. 10. The method of claim 9, wherein the time ranges from about 10 minutes to about 24 hours. 11. The method of claim 9, wherein the temperature ranges from about 20.degree. C. to about 60.degree. C. 12. The method of claim 1, wherein the MALDI matrix is selected from the group consisting of .alpha.-4 cyano hydroxy-cinnamic acid (CHCA), sinnapinic acid, p-nitroaniline, a heavy metal and glycerol. 13. The method of claim 1, wherein the tissue sample is obtained from normal tissue, or abnormal/diseased tissue. 14. The method of claim 13, wherein the diseased tissue is a tumor tissue, tissue from a hyperproliferative disorder other than cancer, or an ischemic tissue. 15. The method of claim 14, wherein the hyperproliferative disorder other than cancer is selected from the group consisting of rheumatoid arthritis, lupus, multiple sclerosis, psoriasis and other autoimmune diseases. 16. The method of claim 14, wherein the tumor tissue is obtained from a benign tumor or a malignant tumor. 17. The method of claim 14, wherein the ischemic tissue is obtained from the brain, spinal cord or other nervous system tissue. 18. The method of claim 14, wherein the ischemic tissue is obtained from the heart or intestinal tract. 19. The method of claim 13, wherein the normal or abnormal/diseased tissue is selected from the group consisting of solid tissue or non-solid tissue. 20. The method of claim 19, wherein the solid tissue is selected from the group consisting of nervous system tissue, cardiac tissue, breast tissue, lung tissue, bladder tissue, gastrointestinal tissue, eyes, bone and tissue from any solid tumor. 21. The method of claim 19, wherein the non-solid tissue is selected from the group consisting of whole blood or isolated blood cells. 22. The method of claim 21, wherein the isolated blood cells are red blood cells or white blood cells. 23. The method of claim 22, wherein the white blood cells are selected from the group consisting of lymphocytes, polymorphonuclear cells (PMNs), monocytes and macrophages. 24. A method for identifying the presence of abnormal or diseased tissue in a subject comprising: a. collecting at least two different tissue samples, one of which is obtained from an area suspected of being diseased or abnormal and the second being normal tissue of the same tissue type; b. treating the tissue samples with a solution of one or more enzymes, or chemicals, capable of digesting the tissue samples into smaller fragments; c. treating the preparation from step b) with a MALDI matrix solution; and d. analyzing the preparation from step c) by DT-MALDI-TOF measurement or DT-MALDI-TOF-TOF measurement, wherein the analyzing comprises comparing the biological molecule content of the at least two different tissue samples, and wherein the biological molecule content of the at least two different tissue samples is compared to a signature map for normal tissue or abnormal or diseased tissue of the same tissue type. 25. The method of claim 24, wherein the signature map of the normal or diseased tissue is obtained from a pre-determined standard or from a known database of proteins isolated and characterized for that tissue and the specific disease of which the subject is suspected of having or at risk for developing. 26. A method for identifying the extent of tumor cell extravasation comprising: a) collecting two or more contiguous tissue samples from a tumor mass and the surrounding tissue; b) treating the tissue samples with a solution of one or more enzymes, or chemicals, capable of digesting the tissue samples into smaller fragments; c) treating the preparation from step b) with a MALDI matrix solution; and d) analyzing the preparation from step c) by DT-MALDI-TOF measurement or DT-MALDI-TOF-TOF measurement, wherein the analyzing comprises comparing the biological molecule content of the two or more contiguous tissue samples, wherein the biological molecule content of the two or more contiguous tissue samples is compared to a signature map for normal tissue or abnormal or diseased tissue of the same tissue type. 27. A method for analyzing the protein content of a cell or bodily fluid sample in situ, comprising: a) collecting a sample of tissue from a subject into a collection device containing a first solution capable of maintaining biological molecule integrity; b) treating the sample with a second solution comprising one or more enzymes, or chemicals, capable of digesting the tissue sample into smaller fragments; c) treating the preparation from step b) with a matrix assisted laser desorption ionization imaging (MALDI) matrix solution; and d) analyzing the preparation from step c) by direct tissue (DT)-matrix assisted laser desorption ionization imaging (MALDI)-time of flight (TOF) measurement or DT-MALDI-TOF-TOF measurement; e) creating a data file utilizing the information from step d); f) entering the data from the data file of step e) into an external database to create a signature map for the tissue from which the data was obtained; and comparing the results from step d) with a signature map for normal tissue, wherein said normal tissue corresponds to the tissue from which the sample was obtained. 28. The method of claim 27, wherein the cell or bodily fluid is selected from the group consisting of urine, serum, plasma, cerebrospinal fluid (CSF), sputum, bone marrow, amniotic fluid and bronchial lavage. 29. A method for determining the presence of a disease in a subject, or for assessing a subject's risk for developing said disease, or for determining a subject's response to a particular therapy for said disease, or for distinguishing between a responder or a non-responder for a particular therapy, the method comprising: a. collecting a first tissue sample from a subject suspected of having a disease or being at risk for developing a disease or being treated for a disease; b. collecting a second cellular or bodily fluid sample from the same subject; c. treating the first tissue sample with a solution comprising one or more enzymes, or chemicals, capable of digesting the first tissue sample into fragments and treating the second cellular or bodily fluid sample with a solution comprising one or more enzymes, or chemicals capable of digesting the second cellular or bodily fluid sample into fragments; d. treating the first tissue sample and the second cellular or bodily fluid sample preparations from step c) with a matrix assisted laser desorption ionization imaging (MALDI) matrix solution; and e. analyzing the preparations from step c) by direct tissue (DT)-matrix assisted laser desorption ionization imaging (MALDI)-time of flight (TOF) measurement or DT-MALDI-TOF-TOF measurement; f. comparing the results from step e) with a signature map for normal tissue, wherein said normal tissue corresponds to the tissue from which the first tissue sample was obtained, and a signature map for normal cells or bodily fluid, wherein the normal cells or bodily fluid correspond to the cells or bodily fluid sample obtained from the subject suspected of having or being at risk for developing said disease, or being treated for said disease. 30. A method for determining the disposition of a new chemical entity or new biological entity in a cell or tissue in vivo, comprising: a) collecting a sample of a tissue or cell from a subject into a first solution capable of maintaining integrity of the tissue or cell sample, prior to treating the subject with a new chemical entity or new biological entity; b) administering a new chemical entity or new biological entity to said subject; c) collecting a series of tissue or cell samples from the subject into a first solution capable of maintaining integrity of the tissue or cell sample at various time points after administering the new chemical entity or new biological entity; d) treating the samples with a second solution comprising one or more enzymes, or chemicals, capable of dissociating the tissue sample or of digesting the dissociated tissue sample into smaller fragments; e) treating the preparation from step d) with a matrix assisted laser desorption ionization imaging (MALDI) matrix solution; and f) analyzing the preparation from step e) by direct tissue (DT)-matrix assisted laser desorption ionization imaging (MALDI)-time of flight (TOF) measurement or DT-MALDI-TOF-TOF measurement; and g) comparing the results from step f) with a series of tissue or cell samples, comparable to the tissue or cell samples collected from the subject, to which has been added either the new chemical entity or new biological entity; h) obtaining a signature map or profile for the new chemical or new biological entity in the series of tissue or cell samples for monitoring the presence or absence of the new chemical entity or new biological entity in the same type of tissue or cell sample from the patient, and i) determining the presence and/or amount of the new chemical entity or new biological entity in said tissue or cell samples. 31. A method for determining the disposition of a new chemical entity or new biological entity in a cell or tissue in situ, comprising: a) collecting a sample of a tissue or cell from a first subject into a first solution capable of maintaining integrity of the tissue or cell sample, prior to treating the first subject with a new chemical entity or new biological entity; b) administering a new chemical entity or new biological entity to said first subject; c) collecting a series of tissue or cell samples from the first subject into a first solution capable of maintaining integrity of the tissue or cell sample at various time points after administering the new chemical entity or new biological entity; d) treating the samples with a second solution comprising one or more enzymes, or chemicals, capable of digesting the tissue sample into smaller fragments; e) treating the preparation from step d) with a matrix assisted laser desorption ionization imaging (MALDI) matrix solution; and f) analyzing the preparation from step e) by direct tissue (DT)-matrix assisted laser desorption ionization imaging (MALDI)-time of flight (TOF) measurement or DT-MALDI-TOF-TOF measurement; and g) comparing the results from step f) with a series of tissue or cell samples collected from a second subject, wherein said second subject has not been administered the new chemical entity or new biological entity, wherein the tissue or cell samples collected from the second subject are identical or comparable to the tissue or cell sample collected from the first subject; h) obtaining a signature map or profile for the new chemical or new biological entity in the series of tissue or cell samples for monitoring the presence or absence of the new chemical entity or new biological entity in the same type of tissue or cell sample from any other subject to be treated in the future with the new chemical entity or new biological entity. 32. A method for determining the intracellular location of a new chemical entity or new biological entity comprising: a) collecting a cell sample, prior to treating the sample with a new chemical entity or new biological entity, into a solution capable of maintaining cellular integrity; b) treating said cell sample with a new chemical entity or new biological entity; c) treating the cell sample from step a) or b) with a second solution comprising one or more enzymes, or chemicals, capable of digesting the cell samples into smaller fragments; d) treating the samples from step c) with a matrix assisted laser desorption ionization imaging (MALDI) matrix solution; and e) analyzing the preparation from step d) by direct tissue (DT)-matrix assisted laser desorption ionization imaging (MALDI)-time of flight (TOF) measurement or DT-MALDI-TOF-TOF measurement; and f) comparing the results from step b) with the results from step a) to determine the presence of the new chemical entity or new biological entity in the cell sample from step b).

Details for Patent 7,714,276

Applicant	Tradename	Biologic Ingredient	Dosage Form	BLA	Approval Date	Patent No.	Expiredate
Smith & Nephew, Inc.	SANTYL	collagenase	Ointment	101995	06/04/1965		2025-09-30