Methods for direct biomolecule identification by matrix-assisted laser desorption ionization (MALDI) mass spectrometry
United States Patent 8525104
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(C) 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.
Inventors:
Pevsner, Paul (New York, NY, US)
Naftolin, Frederick (Woodbridge, CT, US)
Miller, Douglas C. (Belle Mead, NJ, US)
Hillman, Dean (New Hyde Park, NY, US)
Stall, Brian K. (Bedford, NH, US)
Wishnies, Steven M. (Columbia, MD, US)
Application Number:
Publication Date:
09/03/2013
Filing Date:
04/27/2010
Export Citation:
New York University (New York, NY, US)
Primary Class:
Other Classes:
International Classes:
B01D59/44; H01J49/00; H01J49/40
Field of Search:
250/288, 250/281, 435/7.23, 436/66
View Patent Images:
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US Patent References:
7714276Pevsner et al.250/282Murayama et al.356/36Komatsu et al.250/282Caprioli435/7.23Hafeman et al.204/4567109038Scholl et al.436/66Caprioli250/2826756586Caprioli250/282Scholl et al.Caprioli435/7.235808300Caprioli250/288
Other References:
Xu, Dissertation (Ph.D. in Chemistry) Vanderbilt University (2005) pp. 15-16, 18, 34-35, 68, 87-88, 91, 109 and 112.
Chaurand, et al., Toxicologic Pathology (:92-101.
Primary Examiner:
Wells, Nikita
Assistant Examiner:
Smith, Johnnie L.
Attorney, Agent or Firm:
Klauber & Jackson LLC
Parent Case Data:
CROSS-REFERENCE TO RELATED APPLICATIONSThe present application is a continuation of U.S. Ser. No. 11/541,380, filed Sep. 29, 2006 now U.S. Pat. No. 7,714,276 which is a non-provisional application claiming the priority of copending provisional applications U.S. Ser. No. 60/722,206, filed Sep. 30, 2005, and U.S. Ser. No. 60/784,016, filed Mar. 20, 2006, the disclosures of which are incorporated by reference herein in their entireties. Applicants claim the benefits of these applications under 35 U.S.C. §119(e).
A method for analyzing the biological molecule content of a tissue sample obtained from normal tissue or abnormal/diseased tissue in situ, 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, comprising: a) collecting a sample of tissue from a subject into a first solution effective to maintain integrity of the b) treating the sample with a second solution comprising one or more enzymes, or chemicals, effective to dissociate the tissue sample or of digesting the dissociated tissue sample in c) treating the preparation from step b) with a matrix assisted laser desorption ionization imaging (MALDI) 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 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 t 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.
The method of claim 1, wherein the one or more enzymes effective to dissociate the tissue and degrading the tissue into peptide fragments are selected from the group consisting of a collagenase, a lipase and a protease.
The method of claim 1, wherein the time ranges from about 10 minutes to about 24 hours.
The method of claim 1, wherein the temperature ranges from about 20° C. to about 60° C.
The method of claim 1, wherein the MALDI matrix is selected from the group consisting of α-4 cyano hydroxy-cinnamic acid (CHCA), sinnapinic acid, p-nitroaniline, a heavy metal and glycerol.
The method of claim 1, wherein the diseased tissue is a tumor tissue, tissue from a hyperproliferative disorder other than cancer, or an ischemic tissue.
The method of claim 6, wherein the hyperproliferative disorder other than cancer is selected from the group consisting of rheumatoid arthritis, lupus, multiple sclerosis, psoriasis and other autoimmune diseases.
The method of claim 6, wherein the tumor tissue is obtained from a benign tumor or a malignant tumor.
The method of claim 6, wherein the ischemic tissue is obtained from the brain, spinal cord or other nervous system tissue.
The method of claim 6, wherein the ischemic tissue is obtained from the heart or intestinal tract.
The method of claim 1, wherein the normal or abnormal/diseased tissue is selected from the group consisting of solid tissue or non-solid tissue.
The method of claim 11, 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.
The method of claim 11, wherein the non-solid tissue is selected from the group consisting of whole blood or isolated blood cells.
The method of claim 13, wherein the isolated blood cells are red blood cells or white blood cells.
The method of claim 14, wherein the white blood cells are selected from the group consisting of lymphocytes, polymorphonuclear cells (PMNs), monocytes and macrophages.
A method for identifying the presence of abnormal or diseased tissue in a subject comprising: a) collecting at least two different tissue samples obtained from normal tissue or abnormal/diseased tissue, one of which is obtained from an area suspected of being diseased or abnormal and the second being normal tissue of
b) treating the tissue samples with a solution of one or more enzymes, or chemicals, effective to digest the tissue samples in c) treating the preparation from step b) with a MALDI 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, 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.
A method for identifying the extent of tumor cell extravasation comprising: a) collecting two or more contiguous tissue samples obtained from normal tissue or abnormal/diseased tissue from a tumor mass and th b) treating the tissue samples with a solution of one or more enzymes, or chemicals, effective to digest the tissue samples in c) treating the preparation from step b) with a MALDI 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, 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.
Description:
FIELD OF THE INVENTIONThe present invention relates generally to methods for the in situ determination of the types and amount of proteins in a tissue sample by direct tissue matrix associated laser desorption ionization imaging time-of-flight (DT-MALDI-TOF) measurement. The methods allow for direct analysis of tissue samples, without the need for protein extraction and analysis of protein content of the tissue sample by other standard methods, e.g. electrophoretic analysis or other cumbersome or time consuming methods. The methods also allow for the rapid and accurate detection of particular biological molecules in normal and diseased tissue, and also for determining a subject's response to particular therapies.BACKGROUND OF THE INVENTIONTime of flight mass spectrometry from gel isolates or chromatographic columns are accepted means of identifying proteins based on mass and charge (m/z). In 1988, Koichi Tanaka reported that laser irradiation of a mixture of methanol-ethylene glycol-cobalt ultra fine powder (Co UFT) transferred energy to proteins, chymotrypsinogen and lysozyme, generating a vapor/ion-phase of intact macro molecules that could be detected by time of flight (TOF) mass spectrometry (Tanaka, et al., Rapid Commun. Mass Spectrom. 1-153). This demonstration paved the way for using laser desorption ionization mass spectrometry on solid tissue, direct tissue MALDI (DT-MALDI).MALDI (TOF) identifies proteins and peptides as mass charge (m/z) spectral peaks. Further advances, post-source decay (MALDI-PSD) and collision-induced dissociation (MALDI-CID), have become standard methods to identify proteins from cell or tissue homogenates that are responsible for these peaks. (Hansen, et al., Molecular &Cellular Proteomics 9-314; Norris, et al. Anal Chem. 42-6647; Zhang, et al., Am Soc Mass Spectrom 12-1021; Wang, et al. Journal of Proteome Research 97-2403; Vasilescu, et al., Journal of Proteome Research 92-2200; Vandermoere, et al., Oncogene 82-5491; Luo, et al., Molecular Biotechnology 3-244; Le Guezennec, et al., Molecular and Cellular Biology 3-851. However, most of these procedures utilize harsh digestion conditions and protein extraction procedures prior to analysis by MALDI-TOF methods. Matrices have evolved to include small organic molecules and heavy metals that that can be applied to or mixed with the analyte (Tanaka, et al., Rapid Commun. Mass Spectrom. 1-153). The most often used matrices absorb light at 337λ, the wavelength of a nitrogen laser, and thereby facilitate desorption and ionization of adjacent biological materials. Ions of the same charge acquire a si however, their velocity in the ion chamber depends on their respective masses. The ion time of travel to an anode is measured, precisely by the detector and is recorded as a time-mass/charge spectrum with peaks representing proteins in the sample. Based on instrument calibration of standard samples, these values are converted to mass values for National Center for Biotechnology Information (NCBI) database analysis of peptide fragments with Mascot(C), and Blast(C) software. Protein m/z Spectra can be obtained from fluids, cells and tissues of matrix coated protein extraction preparations with Maldi tof and Maldi tof tof. Although protein signature peaks are reported (Norris, et al., Analytical Chemistry 42-6647), there have been no reports of proteins identified directly from cells or tissue using these technologies. Thus, there is a need in the art to provide a more rapid, yet accurate method to identify proteins directly from a tissue sample without a need for further protein extraction of the sample to be followed by protein determination using standard methods such as 1D, 2D or capillary electrophoresis. These needs are addressed by the agents and methods of the present invention.All publications, patent applications, patents and other reference material mentioned are incorporated by reference in their entirety. In addition, the materials, methods and examples are only illustrative and are not intended to be limiting. The citation of references herein shall not be construed as an admission that such is prior art to the present invention.SUMMARY OF THE INVENTIONIn its broadest aspect, the invention provides a method for analyzing the biological molecule content of a tissue or cell in situ, for example, the protein content of a tissue or cell in situ, comprising the use of direct tissue matrix assisted laser desorption ionization imaging time-of flight (DT-MALDI-TOF) measurements, without extracting the proteins from the tissues or cells, whereby such method may disrupt the cellular or tissue architecture and may lead to the production of artifacts, depending on the extraction method used. Particular cell or tissue disruption procedures that result in the production of artifacts include, but are not limited to, procedures such as homogenization, or sonication, or freezing the cells or tissues without first fixing the cells or tissues in a gentle fixative, such as an alcohol, as described herein. These harsh methods of extracting the biological molecule content of a cell or tissue are not utilized by the methods of the present invention. Accordingly, the procedures described herein eliminate the need for such harmful and cell or tissue disruptive extraction methods, and also saves the time needed for carrying out such additional steps, while at the same time diminishing the artifacts observed when such harsh extraction procedures are utilized. More particularly, the invention provides a method to characterize, identify and quantify biological molecules in a mixture without adulterating the results due to freezing (without fixing first), or disrupting the cells by an extraction procedure such as homogenization or sonication or through use of a tissue grinder and sieving mechanism. The biological molecules may be selected from proteins or peptides or fragments thereof, nucleic acids, carbohydrates, lipids, lipoproteins, and the like. The method of the present invention utilizes mass spectrometry for characterization of the accurate mass of a plurality of biological molecules in a mixture, particularly wherein a majority of said biological molecules is characterized, such that a majority of the mixture's components may be identified and/or quantitated. Accordingly, the methods described herein are useful for the diagnosis of a disease or medical condition, and when used alone, or combined with, for example, histological procedures, results in the identification of biomarkers of particular diseases. Such biomarkers may be identified in body cells, or tissues, or may be present in body fluids, such as whole blood, or serum, or plasma, or urine, or cerebrospinal fluid, and the like. In another broad aspect of the invention, the methods and procedures described may be used to follow the cell or tissue distribution of a new chemical entity or therapeutic agent, for which cell or tissue distribution is unknown. For example, the therapeutic agent may be a small organic molecule (synthetic or naturally derived), or a biological molecule used for treating a disease or condition, such as a small interfering nucleic acid molecule, wherein it may be desirable to track its bodily disposition and/or excretion rate, for example. Such a method can then be used in a pre-clinical or clinical setting in order to do, for example, ADME (Absorption, Distribution, Metabolism and Excretion) studies, or toxicity studies.Accordingly, a first aspect of the invention provides a method for analyzing the biological molecule content of a tissue sample in situ, comprising:a) collecting a sample from a subject into a first solution capable of maintaining integrity of theb) 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 inc) treating the preparation from step b) with a matrix assisted laser desorption ionization imaging (MALDI) andd) 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-TOFe) 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 t andg) 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.In another particular aspect, the method provides for an additional step whereby the tissue or cell sample is analyzed by histological means, in order to compare the results to that obtained by DT-MALDI-TOF measurements. Once confirmation of the findings is obtained, preferably after the results have been validated at least three times using both the DT-MALDI-TOF measurement and the histological analysis, and the data is entered into a data file, this histological step may be omitted in future analyses and the biological molecule assessment made using DT-MALDI-TOF alone.In one embodiment, the biological molecule is selected from the group consisting of a protein, a peptide, or fragments thereof, a nucleic acid, including both DNA and RNA, an oligonucleotide or polynucleotide, an antisense molecule, a small interfering nucleic acid molecule, such as a siRNA or shRNA, a carbohydrate, a lipid, a lipoprotein and the like. In another particular embodiment, the chemical used for dissociation of the tissue or cells is formic acid or cyanogen bromide. In another embodiment, the method may be used to study the disposition of a new chemical entity (NCE), such as a small organic molecule (synthetic or naturally derived), or new biological entity (NBE) in vitro or in vivo, whereby pre-clinical or clinical studies are necessary prior to FDA approval of a new chemical or biological entity for therapeutic use. In this manner, the methods of the present invention may be used to track the location of the NCE or NBE in vitro eg. within the cell or tissue. Alternatively, the methods of the present invention may be used to track the disposition of the NCE or NBE within the body, thus providing a new means to perform ADME studies (Absorption, Distribution, Metabolism and Excretion). In another embodiment, the methods of the present invention may prove useful when it is of interest in determining whether two or more therapeutics agents, when delivered to a cell, tissue or organ, result in a toxic effect on the cell. Accordingly, the methods of the present invention allow for potentially determining whether the use of two or more therapeutic agents may be contraindicated, based on the presence or absence of particular biomarkers after administration of the two or more therapeutic agents to a subject. Once such an effect is realized, an in vitro screen may be developed that could assess such an effect on particular cells, or tissues in culture. Thus, in vitro screening for potential toxic profiling of drugs may be envisioned using the methods of the present invention.In one particular embodiment, the mass of enzymatically, or chemically, derived biological molecules is preferably measured or determined to an accuracy of 10 attamols, most preferably to an accuracy of 5 attamols, and particularly preferred to an accuracy of 1 attamol.In another particular embodiment, the tissue or cell sample is prepared using an ultra cryomicrotome. In yet another particular embodiment, the tissue or cell preparation is prepared to allow for resolution of structures at the subcellular level. In yet another particular embodiment, the tissue or cell preparations are sectioned by a microtome to allow for resolution of cell or tissue structures in the range from about 0.05 micron to about 2.0 microns. In yet another particular embodiment, the tissue or cell preparations are sectioned by a microtome to allow for resolution of cell or tissue structures in the range from about 0.1 micron to about 1.5 micron. In yet another particular embodiment, the tissue or cell preparations are sectioned by a microtome to allow for resolution of cell or tissue structures in the range from about 0.5 micron to about 1.0 micron.In another particular embodiment, mass spectrometry may be accomplished by any recognized mass spectrometry method, from a single or tandem mass spectrometer device. The single mass spectrometer may be one of a matrix assisted laser desorption ionization time-of-flight mass spectrometer, electrospray time-of-flight mass spectrometer. The tandem mass spectrometer may be one of a Fourier-transform cyclotron resonance mass spectrometer, an electrospray quadrupole time-of-flight mass spectrometer, a tandem time-of-flight mass spectrometer, a quadrupole ion trap mass spectrometer, and a triple quadrupole mass spectrometer to name only a few.In another particular embodiment, the method provides for both qualitative identification of the proteins in the sample, as well as, a quantitative measurement of the proteins in the sample.In another particular embodiment, the method provides for a level of detection of a protein in a sample in an amount of about 10 attamols, most preferably of about 5 attamols, and particularly preferred of about 1 attamol. As used herein, the term “about” refers to approximately or close to, usually within (i.e., ±) 10% of the given value or quantity.In another particular embodiment, an internal standard is incorporated to provide a means for quantitating the abundance of a protein or peptide, thereby accomplishing absolute quantitation of a protein or peptide in a sample. Relative quantitation of protein/peptide abundances in complex mixtures is accomplished by comparing ion maps generated in different conditions (i.e diseased vs. non-diseased, treated vs. non-treated).In another particular embodiment, a control, or controls of known concentration are included and placed adjacent to the test sample prior to mass spectrometric analysis. In a preferred aspect, the control will be equimolar. In a particular embodiment, the addition of the control(s) will allow for quantitation of the identified proteins.In yet another particular embodiment, the sample may be collected into a collection device, wherein the collection device is selected from the group consisting of a microcapillary pipette, a plastic or glass tube, and a slide for a cellular or tissue sample obtained from a microtime or an ultra cryo microtome.In yet another particular embodiment, the first solution is a buffered solution or an alcohol.In yet another particular embodiment, 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.In yet another particular embodiment, the alcohol is selected from the group consisting of methanol, ethanol, propanol, butanol, isopropyl alcohol and isobutanol.In yet another particular embodiment, 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.In a more particular embodiment, the one or more enzymes may be selected from the group consisting of trypsin, endoprotease-LysC, endoprotease-ArgC, endoprotease-GluC, and chymotrypsin, to name only a few. In another more particular embodiment, the chemical utilized can be cyanogen bromide, or formic acid.In yet another particular embodiment, the one or more enzymes are left in contact for a time and at a temperature sufficient to obtain dissociated tissue and peptide fragments. For example, the time sufficient to obtain dissociated tissue or peptide fragments may range from about 10 minutes to about 2 hours, and in certain cases may range up to about 24 hours. The length of time is dependent upon the type of tissue, the tissue thickness, the temperature used, and the enzyme or chemical used. In yet another particular embodiment, the temperature ranges from about 20° C. to about 60° C.In another embodiment, the peptides identified through use of the methods of the invention are matched to known peptides having the same atomic mass using Mascot(C). Alternatively, they may be identified using a BLAST analysis, the procedure of which is known to those skilled in the art. In another embodiment of the invention, the database of sequences is a database of amino acid sequences of a plurality of proteins. In a further embodiment, the database of sequences is a nucleotide database. Examples of such databases include the National Center for Biological Information (NCBI) database (Pub Med or GenBank), the Human Genome Project (HGP), and PDB (Protein Data Bank). In another particular embodiment, the nucleic acids or proteins identified in a tissue or cellular sample may be compared to any one or more sequences identified by gene chip analysis, such as those provided by Affymetrix. On the other hand, if one desired to compare the tissue or cellular disposition of an unknown drug or therapeutic agent with a known compound, such libraries of compounds may be used. Examples of libraries of compounds that are commercially available include the Available Chemicals Directory (ACD,) the Specs and BioSpecs database, the Maybridge database, and the Chembridge database.In another embodiment, the protein(s) molecular weight, net charge, and mass to name only a few from the plurality of sequences are stored for comparison with experimentally derived protein molecular weights, net charges and mass.The present invention also provides a database of characterized biological components in a mixture, wherein the biological components are described by their ion map, including accurate mass and molecular weight and net charge of the protein or fragments thereof. In particular, the mixture may be described by one or more features including organism, organ source, tissue source, cellular source, treatment conditions, disease condition, etc.In yet another particular embodiment, the MALDI matrix is selected from the group consisting of α.-4 cyano hydroxy-cinnamic acid (CHCA), sinnapinic acid, p-nitroaniline, a heavy metal and glycerol.In yet another particular embodiment, the tissue sample is obtained from normal tissue, or abnormal/diseased tissue.In yet another particular embodiment, the diseased tissue is a tumor tissue, tissue from a hyperproliferative disorder other than cancer, or an ischemic tissue.In yet another particular embodiment, the hyperproliferative disorder other than cancer is selected from the group consisting of rheumatoid arthritis, lupus, multiple sclerosis, psoriasis and other inflammatory or autoimmune diseases.In yet another particular embodiment, the tumor tissue is obtained from a benign tumor or a malignant tumor.In yet another particular embodiment, the ischemic tissue is obtained from the brain, spinal cord or other nervous system tissue.In yet another particular embodiment, the ischemic tissue is obtained from the heart, brain, spinal cord, kidney, liver, or intestinal tract.In yet another particular embodiment, the normal or abnormal/diseased tissue is selected from the group consisting of solid tissue or non-solid tissue.In yet another particular embodiment, 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.In yet another particular embodiment, the non-solid tissue is selected from the group consisting of whole blood or isolated blood cells. In yet another particular embodiment, the isolated blood cells are red blood cells or white blood cells. In yet another particular embodiment, the white blood cells are selected from the group consisting of lymphocytes, polymorphonuclear cells (PMNs), monocytes and macrophages.A second aspect of the invention provides 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 b) treating the tissue samples with a solution of one or more enzymes, or chemicals capable of dissociating the tissue sample and of digesting the dissociated tc) treating the preparation from step b) with a MALDI andd) 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.In one particular embodiment, 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.A third aspect of the invention provides a method for identifying the extent of tumor cell extravasation comprising:a) collecting two or more contiguous tissue samples from a tumor mass and thb) treating the tissue samples with a solution of one or more enzymes or chemicals capable of digesting the tc) treating the preparation from step b) with a MALDI andd) 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.A fourth aspect of the invention provides a method for analyzing the protein content of a cell or bodily fluid sample in situ, comprising:a) collecting a cell or body fluid sample from a subject into a collection device containing a first solution capable of maintainib) treating the sample with a second solution comprising one or more enzymes capable of digesting the sample inc) treating the preparation from step b) with a matrix assisted laser desorption ionization imaging (MALDI) andd) analyzing the preparation from step c) by direct tissue (DT)-matrix assisted laser desorption ionization imaging (MALDI)-time of flight (TOF) measurement.e) comparing the results from step d) with a signature map for normal cells or bodily fluid.In another particular embodiment, the cell or body fluid is selected from the group consisting of urine, serum, plasma, cerebrospinal fluid (CSF), sputum, bone marrow, amniotic fluid, and bronchial lavage.A fifth aspect of the invention provides 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 tb) collecting a second cellular or body fluid sample fc) treating the first tissue sample with a solution comprising one or more enzymes or chemicals capable of digesting the 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 sample ind) 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) ande) 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-TOFf) 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 body fluid, wherein the normal cells or bodily fluid correspond to the cells or body fluid sample obtained from the subject suspected of having or being at risk for developing said disease, or being treated for said disease.In one particular embodiment, the diseases are selected from the group consisting of breast cancer, colon cancer, liver cancer, lung cancer, pancreatic cancer, prostate cancer, gastrointestinal cancer, uterine cancer, cervical cancer, hepatitis and HIV.In another particular embodiment, the biological molecule may be a viral protein, such as, but not limited to those associated with hepatitis virus, human immunodeficiency virus, or human papilloma virus.In yet another particular embodiment, the methods may be used to monitor the effectiveness of therapies by assessing the presence or absence of certain proteins, such as for example, certain tumor antigens such as those noted above. If a therapy is effective, the tumor antigen may decrease in level, whereas if the therapy is not effective, the tumor antigen may show no change or may increase in level.In yet another particular embodiment, the methods of the invention may also be used to assess whether a subject, or a cell, is a “responder” or a “non-responder” as relates to certain therapies or treatments. For example, one may assess whether a subject or a cell is a responder by observing a change for example, a receptor molecule, such as an estrogen receptor, whereby the subject or cell is being treated using a selective estrogen receptor modulator or SERM. A decrease in the growth or proliferation of a tumor cell in vitro or in vivo bearing such estrogen receptor indicates that the subject or cell is responding to such therapy. On the other hand, if a subject or cell is a “non-responder”, one might expect to see no change in proliferation of such a cell bearing the estrogen receptor or one might expect that there actually may be an increase in the number of cells bearing the estrogen receptor. The term “responder”, while it may generally refer to a positive outcome, may also take on a more negative connotation when one is looking at, for example, an untoward reaction or “response” to a drug, such as is often seen in adverse reactions to certain therapies. Thus, a “responder” in this case refers to a person who has responded negatively to a particular therapy. Thus, if a person responds negatively to a drug, the methods of the invention may also be used to identify particular biomarkers that reflect this type of negative response, and this newly identified biomarker can be applied in the future to monitor the response of other patients to such therapy.A sixth aspect of the invention provides a method for determining the disposition of a new chemical entity or new biological entity in a cell or tissue in situ. In one embodiment, the method comprises: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 nb) administering a new chemical entity or new biologicalc) 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 nd) 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 ine) treating the preparation from step d) with a matrix assisted laser desorption ionization imaging (MALDI) andf) 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 andg) 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 nh) 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, andi) determining the presence and/or amount of the new chemical entity or new biological entity in said tissue or cell samples.In yet another embodiment, the method comprises: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 nb) administering a new chemical entity or new biological entity tc) 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 nd) treating the samples with a second solution comprising one or more enzymes, or chemicals, capable of digesting the tissue sample ine) treating the preparation from step d) with a matrix assisted laser desorption ionization imaging (MALDI) andf) 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 andg) 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 frh) 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.In yet another embodiment, the method may be used for drug development purposes, using either primary cells obtained from a subject or using established cell lines in tissue culture. Through use of the methods of the invention, one may be able to not only identify the disposition of the drug within the cell, but also may be able to identify changes in the MALDI-profile of treated cells. For example, one may detect changes in the protein expression pattern resulting from treating the cells with a new drug or biological entity. In order to determine the location of the drug in particular cells or cell compartments, the method comprises:a) collecting a cell sample, prior to treating the sample with a new chemical entity or new biological entity, into a solution capable of maintaininb) treating said cell sample with a new chemical entity or nc) 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 ind) treating the samples from step c) with a matrix assisted laser desorption ionization imaging (MALDI) ande) 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 andf) 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).Other aspects and advantages will become apparent from a review of the ensuing detailed description taken in conjunction with the following illustrative drawings.BRIEF DESCRIPTION OF THE DRAWINGSFIG. 1. T47D breast cancer cell Histone H2B Mass spectrum of Maldi PSD fragmentation peptides from precursor ion AMGIMNSFVNDIFER, (SEQ. ID. NO.1) mass 1742.8.FIG. 2: Stroke model tubulin Mass spectrum of Maldi PSD fragmentation peptides from precursor ion LHFFMPGFAPLTSR, (SEQ. ID. NO. 13) mass 1621.0.FIG. 3A-3B: Mass spectrum of Maldi TOF analysis of lesioned (b) and non lesioned (a) cerebral hemispheres. The spectral peak intensities of the 33 kDa and 43 kDa proteins are the same in both hemispheres. The color marked 65 kDa protein spectral intensity varies with the size of the lesion. It is least in the normal ipsilateral cingulate gyrus region of the lesioned hemisphere (b) and in the non lesioned hemisphere (a). The color marked spectra from the stroke correspond to the colored number overlying the sampled loci in the photomicrograph.FIG. 4: Histologic photomicrograph of the lesioned hemisphere. Note the edema, hyperchromatic nuclei, and paucity of neurons in the lesion compared to the surrounding normal brain. Maldi TOF sampling loci are marked by colored numbers corresponding to the observed spectra in FIG. 3. These numbers represent decreasing lesion size (1-5), and normal ipsilateral cingulate gyrus (6). Note the lesion penumbra (red dots).FIG. 5 provides the results of a histone H2B detailed Mascot search.FIG. 6 provides the results of a histone H2B detailed Mascot search of query peptide K.AMGIMNSFVNDIFER.I (SEQ ID NO:3).FIG. 7 provides the results of a histone H2B detailed Mascot search of query peptide K.LHYCVSCAHNKVVR.N (SEQ ID NO:4).FIG. 8 provides the results of a histone H2B detailed Mascot search of query peptides R.ILSGHRDLFSIELNK.K (SEQ ID NO:5), K.LHYCVSCVIHSKVVR.N (SEQ ID NO:6), R.NFVAVYDGSSSIENLK.A (SEQ ID NO:7).FIG. 9 provides the results of a histone H2B detailed Mascot search of query peptides K.AAGLPSNLVPFERCNR.A (SEQ ID NO:8), SDGDQCASSPCQNGGSCK.D (SEQ ID NO:9), K.HQNILLEVDDFENR.N (SEQ ID NO:10).FIG. 10 provides the results of a histone H2B detailed Mascot search of query peptides K.FEEGENSLLHLKTVK.H (SEQ ID NO:11), K.AGATVYITGRHLDTLR.V (SEQ ID NO:12).FIG. 11 provides the results of a histone H2B Mascot peptide analysis demonstrating MS/MS fragmentation of AMGMNSFVNDIFER (SEQ ID NO: 1).FIG. 12 provides demonstrates the results of a NCBI Blast search of AMGIMNSFVNDIFER (SEQ ID NO:1).FIG. 13 provides the results of a stroke model tubulin detailed Mascot search.FIG. 14 provides the results of a stroke model tubulin detailed Mascot search of query peptide R.LHFEMPGFAPLSTR.G (SEQ ID NO:13).FIG. 15 provides the results of a stroke model tubulin detailed Mascot search of query peptides K.MPDTQVQIFTSPSTR.E (SEQ ID NO:14), R.RHEMMHSGEKPYK.C (SEQ ID NO:15).FIG. 16 provides the results of a stroke model tubulin detailed Mascot search of query peptides MAEAGEGGEDEIQFLR.T (SEQ ID NO:16), K.EERASLLSDLGPCCK.A (SEQ ID NO:17), R.KTSLSASPFEHSSSR.E (SEQ ID NO:18).FIG. 17 provides the results of a stroke model tubulin detailed Mascot search of query peptides R.QELYQEEQAEIIK.L (SEQ ID NO:19), K.IGTSTLLFLVGAWSR.A (SEQ ID NO:20), R.MLQAMGWKEGSGLGR.K (SEQ ID NO:21).FIG. 18 provides the results of a stroke model tubulin detailed Mascot search.DETAILED DESCRIPTION OF THE INVENTIONBefore the present methods and treatment methodology are described, it is to be understood that this invention is not limited to particular methods, and experimental conditions described, as such methods and conditions may vary. It is also to be understood that the terminology used herein is for purposes of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only in the appended claims.As used in this specification and the appended claims, the singular forms “a”, “an”, and “the” include plural references unless the context clearly dictates otherwise. Thus, for example, references to “the method” includes one or more methods, and/or steps of the type described herein and/or which will become apparent to those persons skilled in the art upon reading this disclosure and so forth in their entirety.Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the invention, the preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference I their entireties.DefinitionsThe terms used herein have the meanings recognized and known to those of skill in the art, however, for convenience and completeness, particular terms and their meanings are set forth below.A bioinformatics system is utilized to identify the differences in patterns of biological molecules, for example, polypeptide patterns, in the case and control samples. Patterns can be composed of the relative representation of numerous biological molecules (e.g., polypeptides, small molecules, etc.), the collective profile of which is more important than the presence or absence of any specific entities. By identifying patterns in blood or other patient samples, the methods herein do not only provide the window to the presence of disease and other pathology in some embodiments, but also to the body's ongoing response to the disease or pathologic condition in other embodiments. In a high throughput mode (pipelined system operation), data from a first sample are evaluated in a bio-informatics system at the same time another sample is being processed in a detection device using, for example, a mass spectrometry system.The patterns of biological molecules, eg. polypeptides, present in a sample may be used to identify the disease state of a patient sample in, for example, a diagnostic setting. Samples can be whole blood samples, serum or plasma samples, as well as tissue or bodily fluid samples from a variety of sources that can be used in alternative embodiments. Preferably, though not necessarily, the system used in the diagnostic application is based upon the same technology platform as the platform used to identify the patterns in the first instance. For example, if the platform used to identify the patterns in the first instance is a time of flight (TOF) mass spectrometer, it is preferred that the diagnostic applications of the patterns are run on a time of flight mass spectrometer.The terms “MALDI” and “MALDI-MS” are used interchangeably and refer to matrix assisted laser desorption imaging and matrix assisted laser desorption/ionization mass spectrometry, which entails methods of mass spectrometric analysis which use a laser as a means to desorb, volatize, and ionize an analyte. In MALDI-MS methods, the analyte is contacted with a matrix material to prepare the analyte for analysis. The matrix material absorbs energy from the laser and transfers the energy to the analyte to desorb, volatize, and ionize the analyte, thereby producing ions from the analyte that are then analyzed in the mass spectrometer to yield information about the analyte. “DT-MALDI TOF” refers to direct tissue MALDI time of flight whereby the MALDI procedure is applied directly to a tissue sample, without the need for further extraction of the biological molecule(s) by harsh methods that disrupt the integrity of the tissue or cell. Such methods include homogenization, or sonication to name just a few. “DT-MALDI” or “DT-MALDI-TOF” or “DT-MALDI-TOF-TOF” allows for studying an intact or non-disrupted cell or tissue sample. Such direct tissue analysis allows for fewer artifacts in the sample, thus providing for better accuracy and quantitation of a particular biological molecule.A “matrix” or a “matrix liquid” refers to a material used in MALDI-MS to prepare the sample analyte for analysis. As noted above, this material absorbs energy from the laser and transfers the energy to the analyte to desorb, volatize, and ionize the analyte, thereby producing ions from the analyte that are then analyzed in the mass spectrometer to yield information about the analyte. Samples of such matrix materials or matrix liquids include, but are not limited to sinapinic acid (SA) and derivatives thereof, such as alpha- cinnamic acid and derivatives thereof, such as α-4-cyano hydroxyl cinnamic acid (CHCA); 3,5-dimethoxy-4- 2,5-dihydroxybenzoic acid (DHB); and dithranol. Other examples include heavy metals and glycerol.The term “polypeptide,” “peptide,” “oligopeptide,” or “protein” as used herein refers to any composition that includes two or more amino acids joined together by a peptide bond. It may be appreciated that polypeptides can contain amino acids other than the 20 amino acids commonly referred to as the 20 naturally occurring amino acids. Also, polypeptides can include one or more amino acids, including the terminal amino acids, which are modified by any means known in the art (whether naturally or non-naturally). Examples of polypeptide modifications include e.g., by glycosylation, or other-post-translational modification. Modifications which may be present in polypeptides of the present invention, include, but are not limited to, acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a polynucleotide or polynucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cystine, formation of pyroglutamate, formylation, gamma-carboxylation, glycation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins such as arginylation, and ubiquitination. For purposes herein, polypeptides include, e.g., proteins, peptides, and/or protein fragments.Fragment ion spectra are generated by a process called “collision-induced dissociation” or “CID” in which the amide bonds of a peptide are broken, followed by recording of the fragment ion spectrum. Cleavage of amide bonds results in b-ions (containing the N-terminal) and y-ions (containing the C-terminal). High quality MS/MS spectra of tryptic peptides typically show prominent b and y-ion series. If only these two ions were produced for every amide bond in a 10 residue peptide, the fragment ion spectrum would contain 18 peaks. Ideally, long stable ion series of predominately either the b or y-type would be recovered. In reality, peptide fragmentation is variable and moiety dependent, which leads to gaps and difficulties in analysis. Determining the identity and sequence of a peptide from its MS/MS spectrum is complicated both by the variety and variability of the fragment ions produced. Factors that complicate interpretation of MS/MS spectra are missing ion subsets, internal rearrangements, subsequent fragmentations, and multiple charge states. Also to be considered are the relationship of fragment ion peak intensity to ion series origin and fragment masses, influence of amino acid residues and their derivatives, on neighboring amide bond cleavages, and the link between amino acid composition and neutral loss fragmentation.“Analyzing the protein content” as defined in the present invention refers to the determination of the type or amount of protein in a tissue, cellular or bodily fluid sample using the methods of the present invention.“Collecting” refers to any means or device for acquiring a sample of tissue, cells or other bodily fluid for analysis by the methods of the present invention.“Enzymes capable of dissociating” or “enzymes capable of digesting” a tissue sample or cellular or bodily fluid sample are used interchangeably and refer to any one or more enzymes capable of dissociating the individual cells from the connecting extracellular matrix of a tissue or dissociating the individual cells from a cellular mass or bodily fluid. Dissociation can be obtained using any known procedure, including treatment with enzymes such as trypsin, collagenase, lipase and the like.“Fragment” refers to either a protein or polypeptide comprising an amino acid sequence of at least 5 amino acid residues (preferably, at least 10 amino acid residues, at least 15 amino acid residues, at least 20 amino acid residues, at least 25 amino acid residues, at least 40 amino acid residues, at least 50 amino acid residues, at least 60 amino residues, at least 70 amino acid residues, at least 80 amino acid residues, at least 90 amino acid residues, at least 100 amino acid residues, at least 125 amino acid residues, at least 150 amino acid residues, at least 175 amino acid residues, at least 200 amino acid residues, or at least 250 amino acid residues) of the amino acid sequence of a parent protein or polypeptide, or a nucleic acid comprising a nucleotide sequence of at least 10 base pairs (preferably at least 20 base pairs, at least 30 base pairs, at least 40 base pairs, at least 50 base pairs, at least 50 base pairs, at least 100 base pairs, at least 200 base pairs) of the nucleotide sequence of the parent nucleic acid. Any given fragment may or may not possess a functional activity of the parent nucleic acid or protein or polypeptide. As used herein, the term “fragment” when used in reference to a polypeptide or parent polypeptide is intended to mean any truncated or smaller mass form, corresponding to either carboxyl-terminal, amino-terminal, or both regions, of a reference polypeptide or parent polypeptide. Accordingly, a deletion of a single amino acid from the carboxyl- or amino-terminus is considered a fragment of a parent polypeptide. The term fragment therefore includes deletion of amino acids at the amino- and/or carboxyl-terminus as well as modifications where, for example, an amino acid side chain is removed but the peptide bond remains. A fragment includes a truncated polypeptide that is generated, for example, by polypeptide cleavage using a chemical reagent, enzyme, or energy input. A fragment can result from a sequence-specific or sequence independent cleavage event. Examples of reagents commonly used for cleaving polypeptides include enzymes, for example, proteases, such as thrombin, trypsin, chymotrypsin and the like, and chemicals, such as cyanogen bromide, acid, base, and o-iodobenzoic acid. A fragment can also be generated by a mass spectrometry method including, for example, all types of fragmentation methods and collision induced dissociation (CID). Furthermore, a fragment can also result from multiple cleavage events such that a truncated polypeptide resulting from one cleavage event can be further truncated by additional cleavage events.A “Signature Map” is defined as a multidimensional representation of a protein alone or as a member of a complex tissue or cellular sample. The signature map includes all the physiochemical properties associated with the intact protein as well as that of the identified and qualified polypeptides thereof. Such physiochemical properties include but are not limited to: Intact protein molecular weight, sub-cellular location, protein class, polypeptide molecular weight, net-charge, post-translational modification. In addition a composite of all assigned signature maps could be interpreted as a global representation of the functional forms of proteins in the tissue or cellular sample. A signature map is often represented as a spectral peak of mass spectrometry data.“Time and temperature sufficient to obtain dissociated tissue and peptide fragments” as used herein refers to the amount of time and temperature needed for an enzymatic breakdown of a tissue sample that allows for release of cells from the sample and subsequent enzymatic breakdown of cellular proteins into peptide fragments. A person skilled in the art would be cognizant of how to determine the time and temperature sufficient to obtain such dissociation of tissue into peptide fragments. For example, the conditions necessary for optimization of particular enzyme activity is available from the manufacturers of the particular enzymes. In the manner of the present invention the optimal time for digestion of tissue samples using, for example, trypsin, is about 10 minutes to about 2 hours, and in certain situations, for example, the type of tissue or enzyme and the temperature of incubation, longer times are required, perhaps about 24 hours. The optimal temperature for digestion is about 20° C. to about 60° C.“Tumor tissue” refers to any tissue that harbors cancerous cells. The tumor tissue may be a solid tumor or a non-solid tumor, such as those present in the circulatory system, for example, a leukemia.“Ischemic tissue” refers to tissue that has been deprived of oxygen due to a decrease in the blood supply to a bodily organ, tissue, or cells caused by constriction, obstruction of, or damage to the blood vessels.“Data file” refers to the compilation of data from the inventors' internal studies following assessment of various tissue levels of particular biological molecules. After incorporating all of the data acquired following various analyses from different types of cells or tissues, both normal and diseased or abnormal, the data files are now compared to a series of biomolecules identified in an external “database of proteins”, for example, the NCBI database.“Tumor cell extravasation” refers to the process whereby a cancer cell exits a blood vessel or lymphatic vessel.“Body fluid” refers to any non-solid sample obtained from a subject or patient, for example, blood, serum, plasma, urine, cerebrospinal fluid, amniotic fluid, tears and the like.“Responders” as used herein refers to a subject or patient who demonstrates a positive outcome from a particular treatment regimen or therapy.“Non-responders” as used herein refers to a subject or patient who does not demonstrate a positive outcome from a particular treatment regimen or therapy.“Subject” or “patient” refers to a mammal, preferably a human, in need of treatment for a condition, disorder or disease.“Post Source Decay” refers to a technique specific to reflectron time-of-flight mass spectrometers where product ions of metastable transitions or collision-induced dissociations generated in the drift tube prior to entering the reflectron are m/z separated to yield product ion spectra.The term “in situ” as used herein refers to the examination of cells or tissues directly.The phrase “maintaining biological molecule integrity” or “maintaining protein integrity” are used interchangeably and both refer to the use of solutions that either prevent cross-linking of proteins or prevent tissue autolysis. Alternatively, in order to maintain biological molecule integrity or protein integrity, it is important to avoid the use of known denaturants such as, but not limited to, formalin, formaldehyde, or glutaraldehyde. The use of buffered alcohols is an acceptable means for maintaining biological molecule or protein integrity. Another means of maintaining biological molecule integrity is through the use of a cryoprotectant. A “cryoprotectant” is a compound that prevents cell damage during freezing and thawing processes. Cryoprotectants are agents with high water solubility and low toxicity. Examples of cryoprotectant agents are glycerol, DMSO, sugars, dextran, ethylene glycol, methylene glycol, polyvinyl pyrolidone and hydroxyethyl starch.The terms “biological molecule” or “biological molecules” or “biomolecules” refer to any one or more of the following constituents of cells or tissues: proteins, polypeptides, or peptide fragments thereof, nucleic acids, including DNA or RNA or an oligonucleotide or fragment or a complement thereof, carbohydrates, lipids, lipoproteins and the like. A biological molecule may also refer to a metabolite, such as, but not limited to, small molecule metabolites, such as sugars, folic acid, uric acid, lactic acid, or glutathione.“Chemicals capable of dissociating” a tissue sample or cellular or bodily fluid sample refers to any one or more chemicals capable of dissociating the individual cells from the connecting extracellular matrix of a tissue or dissociating the individual cells from a cellular mass or bodily fluid. Examples of such chemicals include cyanogens bromide or formic acid.A “mechanical means of dissociating” a tissue sample or cellular or bodily fluid sample refers to the use of any one or more mechanical means capable of dissociating the individual cells from the connecting extracellular matrix of a tissue or dissociating the individual cells from a cellular mass or bodily fluid. Examples of such mechanical means include the use of ultrasound, for example, sonication, or the use of a tissue homogenizer or grinder and a sieving mechanism to separate out the individual cellular components from a tissue or bodily fluid.The term “extracting” or “extraction” refers to the use of a biological, chemical, physical or mechanical means of isolating a biological molecule before analysis by any of the MALDI procedures described herein. Certain methods of extracting are much harsher than others, and these generally include, but are not limited to, procedures such as sonication or homogenization. Moreover, the use of such mechanical means for the isolation of a biological molecule of interest may lead to disruption in the cell or tissue architecture with the production of artifacts. The methods of the present invention avoid the use of such extraction procedures in order to reduce the possibility of introducing such artifacts into the samples under analysis. Another means of extracting a biological molecule is through use of a freezing procedure. However, if one attempts extracting by freezing, it is important to maintain the biological molecule integrity by the prior addition of a cryoprotective agent, such as those used cryoprotectives used in the present invention. Freezing without cryoprotection also leads to disruption of the cellular or tissue architecture and the production of artifacts. Such cryoprotective agents include buffered and non-buffered alcohols, sucrose and other agents such as methylene glycol andGeneral DescriptionIn its broadest aspect, the present invention provides a method to characterize, identify and quantify biological molecules in a mixture. The method of the present invention utilizes direct tissue mass spectrometry for characterization of the accurate mass of a plurality of biological molecules in a mixture, particularly wherein a majority of said biological molecules is characterized, such that a majority of the mixture's components may be identified and/or quantitated.All prior methods of tissue protein analysis required extraction of the proteins from various homogenized or otherwise prepared specimens. As noted above, this allows for the generation of many artifacts within the sample and has other practical problems associated with it. The method described herein provides for direct tissue analysis of biological molecules, such as proteins, by a unique method of preparation and examination of the tissue that will allow direct, rapid, simultaneous analysis of a wide variety and large number of diverse biological molecules, including proteins from normal or abnormal tissues or cells. This method circumvents the need for specialized reagents, such as antibodies, that are dependent on specialized portions of the molecules such as epitopes. This method will thereby characterize the au natural protein composition of cells, fluids and tissues for the purpose of identification of a biomolecule, such as a protein, that could serve as a biomarker for particular diseases or conditions. For example, the use of the methods of the invention for the identification of a particular biomolecule in a diseased tissue or cell sample, which is absent in a sample of normal or non-diseased tissue or cells, would qualify the biomolecule as a biomarker for that disease or condition, such as a cancerous condition. The methods described herein reveal the state of the proteins in their natural environment. This invention, in conjunction with special techniques, such as protein crystallography and electron microscopy, reveals complex molecular structures and configurations (folding, misfolding, and fusion proteins). The present invention reveals changes in protein expression that will allow evaluation of the clinical risk of disease, effect of environmental agents, progress of differentiation and disease or other clinical pathologic conditions. This method is not dependent on the formation of derivatives or other surrogates to identify molecular structure. The method identifies molecular structure from tissues by their molecular mass. The method is independent of s}