Patent Grant
7728287
1. Field of Endeavor
The present invention relates to mapping of cells and tissue and more particularly to imaging mass spectrometry with mass tags.
2. State of Technology
U.S. Pat. No. 5,808,300 for a method and apparatus for imaging biological samples with MALDI MS, issued Sep. 15, 1998 to Richard M. Caprioli and assigned to Board of Regents, The University of Texas System provides the following state of technology information: “The combination of capillary electrophoresis (CE) and mass spectrometry (MS) provides an effective technique for the analysis of femtomole/attomole amounts of proteins and peptides. The low load levels and high separation efficiency of capillary electrophoresis are well suited to the mass measurement capability and high sensitivity of mass spectrometry. A considerable amount of work has been published using electrospray mass spectrometry for on-line coupling to capillary electrophoresis.”
U.S. Pat. No. 6,756,586 for methods and apparatus for analyzing biological samples by mass spectrometry, issued Jun. 29, 2004 to Richard M. Caprioli and assigned to Vanderbilt University provides the following state of technology information: “A specimen is generated, which may include an energy absorbent matrix. The specimen is struck with laser beams such that the specimen releases proteins. The atomic mass of the released proteins over a range of atomic masses is measured. An atomic mass window of interest within the range of atomic masses is analyzed to determine the spatial arrangement of specific proteins within the sample, and those specific proteins are identified as a function of the spatial arrangement. By analyzing the proteins, one may monitor and classify disease within a sample.”
Features and advantages of the present invention will become apparent from the following description. Applicants are providing this description, which includes drawings and examples of specific embodiments, to give a broad representation of the invention. Various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this description and by practice of the invention. The scope of the invention is not intended to be limited to the particular forms disclosed and the invention covers all modifications, equivalents, and alternatives falling within the spirit and scope of the invention as defined by the claims.
Mass spectrometry techniques are highly sensitive tools for chemical analysis of a wide range of materials. The applications of mass spectrometry for biological cell analyses are just beginning. Applicants' studies show the utility of ToF-SIMS analysis and multivariate statistical techniques for characterizing the origin, developmental stage and disease state of single cells. These methods can detect physical, chemical, or radiation damage in individual cells, with the capability of determining the molecules that are the basis of changes detected. The methods enable new discoveries to be made by chemically analyzing single cells.
The present invention provides a method of analyzing biological material. The method includes exposing the biological material to a recognition element, exposing the biological material to a mass tag element, directing an ion beam of a mass spectrometer to the biological material, interrogating at least one region of interest area from the biological material and producing data, and analyzing the data to provide information about the biological material. In one embodiment the step of analyzing the biological material includes obtaining known data and comparing said data with said know data. In another embodiment the step of analyzing the biological material includes distributing the data in plots indicating measures of similarity.
The present invention can be used with broad-based mass spectrometry techniques such as time-of-flight secondary ion mass spectrometry (ToF-SIMS), and matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) to understand the intracellular localization of tagged molecules and pathway fluxes. Examples of specific uses are detecting markets for normal and cancerous cells, identifying markers for physical, chemical or radiation damage, understanding metabolite fluxes in single cells or categorizing the tissue of origin of a cell.
The present invention can be used for medical diagnostic and prognostic applications and for fundamental studies of biological processes. The methods involve individual eukaryotic and prokaryotic cells or multi-cellular tissues. The technology will be especially applicable to problems that require localization of known targets and pathways with cells or tissues. This method will allow 10-1000 molecular species to be evaluated for classification of cancers for diagnosis and treatment (multiplex analysis). Single cell or tissue analysis can be used for mass spectrometry-based medical diagnostics and basic and applied research. The present invention can be used for projects in cancer detection, stem cell development, drug studies and environmental analyses.
The invention is susceptible to modifications and alternative forms. Specific embodiments are shown by way of example. It is to be understood that the invention is not limited to the particular forms disclosed. The invention covers all modifications, equivalents, and alternatives falling within the spirit and scope of the invention as defined by the claims.
The accompanying drawings, which are incorporated into and constitute a part of the specification, illustrate specific embodiments of the invention and, together with the general description of the invention given above, and the detailed description of the specific embodiments, serve to explain the principles of the invention.
Referring to the drawings, to the following detailed description, and to incorporated materials, detailed information about the invention is provided including the description of specific embodiments. The detailed description serves to explain the principles of the invention. The invention is susceptible to modifications and alternative forms. The invention is not limited to the particular forms disclosed. The invention covers all modifications, equivalents, and alternatives falling within the spirit and scope of the invention as defined by the claims.
Referring now to the drawings and in particular to
In the method 100, biological materials are exposed to detection molecules consisting of a recognition element and a mass tag element. These two elements are cleavable. The recognition element binds to a specific chemical or protein structure. When the material is analyzed, the mass tags are released by the mass spectrometer ion beam, by photolysis, or other means to make the mass tag detectable by the instrument, thus localizing the distribution and quantity of the chemical or protein identified by the detection element.
Referring again to
The biological materials, fluids, cells or tissues, are placed on chips of silicon or other suitable material. Samples are analyzed directly or the cell contents exposed by crushing, freeze-fracturing or other methods. Samples are placed in an imaging mass spectrometer such as a ToF-SIMS, or ToF-SIMS/MALDI.
Step 103 uses an ion beam of a mass spectrometer directed to the biological material. The ion beam is a finely focused energetic primary-ion beam of a time-of-flight secondary ion mass spectrometer. In another embodiment the ion beam is an ion beam of a matrix-assisted laser desorption/ionization mass spectrometer.
In the method 100B, biological materials are exposed to detection molecules consisting of a recognition element and a mass tag element. These two elements are cleavable. The recognition element binds to a specific chemical or protein structure. When the material is analyzed, the mass tags are released by the mass spectrometer ion beam, by photolysis, or other means to make the mass tag detectable by the instrument, thus localizing the distribution and quantity of the chemical or protein identified by the detection element.
Referring again to
The biological materials, fluids, cells or tissues, are placed on chips of silicon or other suitable material. Samples are analyzed directly or the cell contents exposed by crushing, freeze-fracturing or other methods. Samples are placed in an imaging mass spectrometer such as a ToF-SIMS, or ToF-SIMS/MALDI.
Step 103B uses an ion beam of a mass spectrometer directed to the biological material. The ion is a finely focused energetic primary-ion beam of a time-of-flight secondary ion mass spectrometer. In another embodiment the ion beam is an ion beam of a matrix-assisted laser desorption/ionization mass spectrometer.
Referring now to
Use of Ga and Au ions to chemically map the surface of cells or the interior of crushed or fractured cells can be quite useful in telling one cell from another, but understanding what protein or expressed gene is responsible for the difference requires more specific analysis. This is why using the same imaging technology but putting specific masses attached to ligands that can recognize DNA sequences (oligos) or specific proteins (antibodies) can give the method specificity. In addition, multiplexing 10-100 of these mass tagged detectors in the same cell would allow analysis of many macromolecules in a pathway or system at the same time. No method exists today that can do this at the single cell level and also image the result.
In the method 100C, biological materials are exposed to detection molecules consisting of a recognition element and a mass tag element. These two elements are cleavable. The recognition element binds to a specific chemical or protein structure. When the material is analyzed, the mass tags are released by the mass spectrometer ion beam, by photolysis, or other means to make the mass tag detectable by the instrument, thus localizing the distribution and quantity of the chemical or protein identified by the detection element.
Referring again to
In steps 105C, 106C and 107C a recognition element (Ab) and a mass tag element (Mass 224) are connected by a cleavable linker (UV Cleavable linker). This provides the recognition element and a mass tag element connected by a cleavable linker (Ab-Mass 224) shown as block 108C.
In step 109C the cleavable linker (Ab-Mass 224) is cleaved. For example, exposure to ultraviolet light cleaves the cleavable linker.
In step 103C an ion beam is directed to the biological material. For example, in step 103C an ion beam of a mass spectrometer is directed to the biological material. In step 103C at least one region of interest area from the biological material is interrogated and data is produced.
In step 104C the data is analyzed to provide information about the biological material. For example, the data analysis spectra includes a mass tag from an individual cell. In step 104C the Time-of-Flight Secondary Ion Mass Spectrometry (ToF-SIMS) produces a chemical map of the surface of a biological sample.
Referring now to
The mass spectra from region of interest 202 is exported to statistical analysis software, and the data set for each cell or region are distributed in plots indicating measures of similarity. Single cell mass spectral data sets can be compared to known samples to identify a cells' tissue of origin, understand the cells metabolic state, or predict progression to disease state.
These results will be enhanced by the ability to image proteins and mRNA in the same environment as metabolites. Single cells and tissue specimens will be the main source of material. This has never been done before and is a concept at this time. 10 to 1000 molecular species will be measured at once in the same cell (multiplex analysis).
Referring now to
These results will be enhanced by the ability to image proteins and mRNA in the same environment as metabolites. Single cells and tissue specimens will be the main source of material. This has never been done before and is a concept at this time. 10 to 1000 molecular species will be measured at once in the same cell (multiplex analysis).
Referring now to
In the series of figures
The article, “Chemical and biological differentiation of three human breast cancer cell types using time-of-flight secondary ion mass spectrometry (TOF-SIMS)” by K. S. Kulp, E. S. F. Berman, M. G. Knize, D. L. Shattuck, E. J. Nelson, L. Wu, J. L. Montgomery, J. S. Felton and K. J. Wu (2006), in Analytical Chemistry, 78:6351-6358. (Web Release Date: May 5, 2006), includes the statements, “We use Time-of-Flight Secondary Ion Mass Spectrometry (TOF-SIMS) to image and classify individual cells based on their characteristic mass spectra. Using statistical data reduction on the large data sets generated during TOF-SIMS analysis, similar biological materials can be differentiated based on a combination of small changes in protein expression, metabolic activity and cell structure. We apply this powerful technique to image and differentiate three carcinoma-derived human breast cancer cell lines (MCF-7, T47D and MDA-MB-231). In homogenized cells, we show the ability to differentiate the cell types as well as cellular compartments (cytosol, nuclear and membrane). These studies illustrate the capacity of TOF-SIMS to characterize individual cells by chemical composition, which could ultimately be applied to detect and identify single aberrant cells within a normal cell population. Ultimately, we anticipate characterizing rare chemical changes that may provide clues to single cell progression within carcinogenic and metastatic pathways.” The article, “Chemical and biological differentiation of three human breast cancer cell types using time-of-flight secondary ion mass spectrometry (TOF-SIMS)” by K. S. Kulp, E. S. F. Berman, M. G. Knize, D. L. Shattuck, E. J. Nelson, L. Wu, J. L. Montgomery, J. S. Felton and K. J. Wu (2006), in Analytical Chemistry, 78:6351-6358. (Web Release Date: May 5, 2006), is incorporated herein by this reference.
The ion beam 500 in this embodiment is a finely focused energetic primary-ion beam of a time-of-flight secondary ion mass spectrometer that is directed to the small groups of cells or the single cell and tissues or surrounding materials. At least one region of interest is interrogated. At least one region of interest can be an area from individual cells, tissues or surrounding materials. Time-of-Flight Secondary Ion Mass Spectrometry (ToF-SIMS) is a surface sensitive technique that allows the detection and localization of the chemical composition of sample surfaces. The instrument uses a finely focused (˜300 nm), pulsed primary ion beam 500 to desorb and ionize molecular species from a sample surface.
The mass spectra from region of interest is exported to statistical analysis software, and the data set for each cell or region are distributed in plots indicating measures of similarity. Single cell mass spectral data sets can be compared to known samples to identify a cells' tissue of origin, understand the cells metabolic state, or predict progression to disease state.
These results will be enhanced by the ability to image proteins and mRNA in the same environment as metabolites. Single cells and tissue specimens will be the main source of material. This has never been done before and is a concept at this time. 10 to 1000 molecular species will be measured at once in the same cell (multiplex analysis).
Referring now to
The mass spectra from region of interest is exported to statistical analysis software, and the data set for each cell or region are distributed in plots indicating measures of similarity. Single cell mass spectral data sets can be compared to known samples to identify a cells' tissue of origin, understand the cells metabolic state, or predict progression to disease state.
Rat mammary cell lines, differing in metastatic potential, are well-separated by PCA, but what molecules are responsible for the differences? Mass tag technology can answer that question. Rat mammary adenocarcinoma cell lines that were derived from the same tumor. MTLn3 cells have the potential to cause distant metastases, MTC cells do not; model for metastasis. MCF-7 is a human breast cancer cell line. It is possible to tell which proteins determine the malignancy of MTLn3 and not MTC.
Referring now to
One of the two forms of these reagents has an antibody connected by a UV linker to molecule with specific mass. This allows identification of that antibody binding specific from others in the multiplex reagent. UV cleaves the tag away from the antibody which is hundreds of times larger. This method can be used on individual cells or paraffin embedded tissues. It will contribute to cancer prognosis and drug effectiveness determinations.
Referring now to
While the invention may be susceptible to various modifications and alternative forms, specific embodiments have been shown by way of example in the drawings and have been described in detail herein. However, it should be understood that the invention is not intended to be limited to the particular forms disclosed. Rather, the invention is to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the invention as defined by the following appended claims.
The United States Government has rights in this invention pursuant to Contract No. W-7405-ENG-48 between the United States Department of Energy and the University of California for the operation of Lawrence Livermore National Laboratory.
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