# Patent

## Title
Biomarkers of ionizing radiation

## Abstract

The present invention provides novel radiation associated markers. The radiation associated markers may be one or more of albumin, LTGF-β, or any protein or peptide listed in any one of Tables 1, 2, 3, 4, 5, and 6 provided herein. The present invention also provides methods of assessing exposure to ionizing radiation by determining the presence of one or more radiation associated markers. The methods may optionally include quantifying one or more of the radiation associated markers. The methods may further include comparing the amount of one or more radiation associated markers in the sample determined to be present in the sample with either (i) the amount determined for temporally matched, normal samples or (ii) the amount determined for samples obtained from individuals or subjects that have not been exposed to an elevated level of ionizing radiation.

## Background
CROSS REFERENCE TO RELATED APPLICATIONS
This application is a continuation of U.S. Ser. No. 12/387,508 filed May 4, 2009 now U.S. Pat. No. 8,269,163 which claims priority to U.S. provisional patent application Ser. No. 61/126,246, filed on May 3, 2008, the disclosures of which are herein incorporated by reference in their entirety.


FIELD OF THE INVENTION
This invention relates to new protein biomarkers that may be present after exposure to ionizing radiation and methods of assessing exposure to ionizing radiation as well as diagnostic tests and kits for evaluating exposure to ionizing radiation.
BACKGROUND OF THE INVENTION
Recent work has identified a transcription factor, nuclear factor KAPPA B (NF-KB) which induces the TNF-α encoding gene and activates the cyclooxygenase-2 (COX-2) pathway. At 24 hours post irradiation HIF-1a and COX-2 protein levels were increased. In addition to its well established DNA-damage effects, ionizing radiation induces cell death, and radiation-induced activation of acid sphingomyelinases (ASMases) and the generation of ceramide. Ceramide is generated from sphingomyeline by the action of a neutral or ASMase or by de novo synthesis coordinated through the enzyme ceramidesynthase. Once generated, ceramide may serve as a second messenger molecule in signaling responses to physiologic or environmental stimuli, or it may be converted to a variety of structural or effector molecules. With a single dose of 3 Gy, there is activation of protein kinase B/AKT (PKB/AKT) signaling. Within minutes of irradiation, phosphorylation of the serine/threonin protein kinase PKB/AKT at serine-residue 473 appears. This activation of PKB/AKT contributes to inhibit glycogen synthase kinase-3beta (GSK3beta), which has a clear inhibitory role in endothelial cell survival.
One problem first responders face in the event of a nuclear disaster is to rapidly and efficiently identify victims that need medical treatment and determine what level of treatment is appropriate. It is a priority to minimize time to diagnosis to identify patient treatment needs, minimize the time required for emergency technicians (ET) provide diagnosis, minimize the number of interactions with the patient, minimize system stockpile cost, minimize the dependence on infrastructure (communications, transport, etc.), minimize the needs for trained personnel to administer tests, and uniquely identify patient and diagnostic results.
Simple, quick test strips that change color, like the ones used for sugar or albumin by diabetics, are currently available. They are used without medical or other highly trained personal or expensive equipment. It would be highly desirable to provide such a simple test strip that changes color that could be used as evidence of ionizing radiation (IR) exposure. A positive test that could be used to identify a person who has been exposed to radioactive material has many applications.
SUMMARY OF THE INVENTION
In a first aspect, the present invention provides novel radiation associated markers. The radiation associated markers may be one or more of albumin, LTGF-β, or any protein or peptide listed in any one of Tables 1, 2, 3, 4, 5, and 6 provided herein.
In a second aspect, the present invention provides methods of assessing exposure to ionizing radiation by determining the presence of one or more radiation associated markers. The radiation associated markers may be one or more of albumin, LTGF-β, or any protein or peptide listed in any one of Tables 1, 2, 3, 4, 5, and 6 provided herein.
The sample used for determining the presence of one or more radiation associated markers may be, for instance, saliva, a buccal swab, amniotic fluid, plasma, serum, urine or blood. The one or more radiation associated markers may be identified by many methods well known to those of skill in the art including Matrix assisted laser desorption ionization (MALDI) mass spectrometry. The radiation associated markers may also be identified by contacting the sample with an antibody which specifically binds to the radiation associated marker under conditions permitting formation of a complex between the antibody and the radiation associated marker, and optionally measuring the amount of complexes formed, thereby determining the amount of the radiation associated marker.
The methods may optionally include quantifying one or more of the radiation associated markers. The methods may further include comparing the amount of one or more radiation associated markers in the sample determined to be present in the sample with either (i) the amount determined for temporally matched, normal samples or (ii) the amount determined for samples obtained from individuals or subjects that have not been exposed to an elevated level of ionizing radiation. The relative absence of one or more of the radiation associated markers in the sample indicates that the individual has not been exposed to an elevated level of ionizing radiation, and the relative abundance of one or more of the radiation associated markers in the sample indicates that the individual has been exposed to an elevated level of ionizing radiation. In some embodiments, one or more of the radiation associated markers are not present in detectable levels in normal tissue unaffected by elevated levels of ionizing radiation at all. In other embodiments, one or more of the radiation associated markers is present in levels that are 10%, 20%, 30%, 50%, 75% or more greater than the amount of the corresponding radiation associated marker in normal tissue unaffected by elevated levels of ionizing radiation at all. In still other embodiments, one or more of the radiation associated markers is present in levels that are 2×, 3×, 4×, 5×, 10×, 25×, 50×, 100×, 1,000×, or even 10,000× or more greater than the amount of the corresponding radiation associated marker in normal tissue unaffected by elevated levels of ionizing radiation at all.
In some embodiments, the elevated levels of ionizing radiation may be 10%, 20%, 30%, 50%, 75% or more greater than the amount of ionizing radiation experienced normally from environmental sources. In still other embodiments, the elevated levels of ionizing radiation may be 2×, 3×, 4×, 5×, 10×, 25×, 50×, 100×, 500×, 1,000×, 5,000×, 10,000× 100,000×, 1,000,000× or more greater than the amount of ionizing radiation experienced normally from environmental sources.
The methods of the present invention are applicable to determining the relative amount of ionizing radiation to which an individual has been exposed. Likewise, the methods of the present invention are applicable to determining the relative amount of damage caused by ionizing radiation to an individual. The relative amount of ionizing radiation to which an individual has been exposed and the relative amount of damage caused by ionizing radiation to an individual may be determined, for instance, by quantifying the amount of one or more radiation associated marker present in a tissue or by determining the presence of one or more radiation associated marker that is not present in a tissue until a particular threshold of ionizing radiation is experience or until a particular threshold of damage to a tissue is crossed.
In a third aspect, the present invention further provides a method of predicting outcome in a subject after exposure to elevated levels of ionizing radiation. In some embodiments, it is a method of predicting the likelihood of surviving long term after exposure to elevated ionizing radiation. In other embodiments, it is a method of predicting the relative amount of damage caused to an individual or to a tissue by exposure to elevated ionizing radiation comprising determining the presence or the relative presence as compared to normal samples or normal subjects of one or more radiation associated markers. One, two, three, or more of the radiation associated markers may be present in the sample and may be identified. One, two, three, or more of the radiation associated markers may be present in the sample and may be elevated in comparison to samples obtained from individuals that have not been exposed to elevated levels of ionizing radiation.
The sample used for determining the presence of one or more radiation associated markers may be, for instance, saliva, plasma, serum, urine or blood. The one or more radiation associated markers may be identified by many methods well known to those of skill in the art including Matrix assisted laser desorption ionization (MALDI) mass spectrometry. The radiation associated markers may also be identified by contacting the sample with an antibody which specifically binds to the radiation associated marker under conditions permitting formation of a complex between the antibody and the radiation associated marker, and optionally measuring the amount of complexes formed, thereby determining the amount of the radiation associated marker in the sample.
The methods may optionally include quantifying one or more of the radiation associated markers. The methods may further include comparing the amount of radiation associated markers in the sample determined to be present in the sample with either (i) the amount determined for temporally matched, normal samples or (ii) the amount determined for samples obtained from individuals that have not been exposed to elevated levels of ionizing radiation. The relative absence of one or more of the radiation associated markers in the sample indicates that the likelihood of significant damage, death, illness or medical complications is relatively low, and the relative abundance of one or more of the radiation associated markers in the sample indicates that the likelihood of significant damage, death, illness or medical complications is relatively high.
The invention provides methods for determining the relative likelihood of significant damage, death, illness or medical complications comprising the steps of: (a) obtaining a biological sample from an individual; (b) measuring an amount of one or more radiation associated markers present in the biological sample; and (c) comparing the amount of one or more radiation associated markers with a predetermined value, whereby the amount of radiation associated markers relative to the predetermined value indicates the likelihood of significant damage, death, illness or medical complications or the relative likelihood of a positive or negative medical outcome.
The methods of the invention can be used alone or in combination with any known test for determining the relative likelihood of significant damage, death, illness or medical complications or for determining the prognosis of exposure to ionizing radiation, including, but not limited to, X-Ray, ultrasound, CAT scan, and other blood marker analysis. The methods of the invention may be used to screen a biological sample collected at any time either before or after a first exposure to elevated levels of ionizing radiation has occurred.
In a fourth aspect, the present invention further provides a method of determining the amount of radiation therapy that has been delivered to a particular tissue. In other embodiments, it is a method of predicting the relative amount of damage caused to a particular tissue by exposure to elevated ionizing radiation from radiation therapy comprising determining the presence or the relative presence as compared to normal samples or normal subjects of one or more radiation associated markers. One, two, three, or more of the radiation associated markers may be present in the tissue and may be identified. One, two, three, or more of the radiation associated markers may be present in the tissue and may be elevated in comparison to tissues that have not received radiation therapy.
In a fifth aspect, the present invention provides a kit for assessing the likelihood of significant damage, death, illness or medical complications post exposure to elevated levels of ionizing radiation by determining the presence or absence or by quantifying the amount of one or more radiation associated markers. The kits may contain biomarker identification and bioassay systems that address both the problem of a very rapid and inexpensive Point of Care (POC) diagnostic determination of radiation exposure and of quantitative High Throughput (FIT) diagnostic determination of radiation exposure. The kits and bioassay systems are useful as a means for making a rapid diagnosis and a quantitative high throughput diagnosis. The kits and bioassay systems utilize biochemistry that serves as the basis for assay systems based on one or more biomarkers indicative of a dose or ionizing radiation received. Immunoassays may be provided to quantitatively measure radiation dose. These immunoassays may be incorporated into commercial diagnostic systems using both an established strip test technology to provide a >2 Gy screening diagnosis for quick triage and microfluidics technology that provides a low cost high throughput quantitation capability to indicate dosing level at +/−0.5 Gy to determine the course of treatment to be received. Examples of such microfluidics devices include a handheld microfluidic reader available from Claros Diagnostics, Inc.
The kits and bioassays use identified biomarkers. Commercial antibodies may be obtained or immunoassays developed to provide a quantitative measure of the biomarkers. The commercial antibodies or immunoassays may be embedded into a test strip using established technology. These test strips may provide, for instance, a 0-5 or 0-2 Gy quantitative screening diagnosis for quick triage. In some embodiments, the test strips may change color to provide a useful gauge of the amount of elevated ionizing radiation to which a tissue or an individual has been exposed or to gauge the amount of damage a tissue or an individual has experienced.



BRIEF DESCRIPTION OF THE FIGURES
FIG. 1 demonstrates the presence of radiation associated biomarkers post exposure to elevated levels of ionizing radiation.
FIG. 2 demonstrates the presence of radiation associated biomarkers post exposure to elevated levels of ionizing radiation.
FIG. 3 represents a MALDI spectrum, linear mode, control, 15 minutes post 3 Gy, and 30 minute post 3 Gy. Note the protein peaks 63870.32 Da at 15 minutes, and 72449.25 at 30 minutes. These proteins can be tentatively identified with bioinformatics in the NCBInr database. The tip of the 63870.32 peak is superimposed on the bottom of the 30 minute spectrum.
FIG. 4 represents MALDI, reflectron mode, control, bottom; 15 minutes post 3 Gy, center; and 30 minute post 3 Gy, top. Note 955.87 Da and 1184.12 Da peptides that appear at 15 minutes post 3 Gy, and the 939.07 Da peptide that appears 30 minute post 3 Gy.
FIG. 5 represents a MALDI image of murine tongue one hour post 1 Gy TBI, longitudinal section. Note peripheral foci of Albumin (Red in image).
FIG. 6 depicts the histopathology of murine tongue one hour post 1 Gy TBI, longitudinal section. Note minimal destructive changes in the epithelial cornified spicule layer and edema in the basal region.
FIG. 7 represents a MALDI image. Note the marked increase in peripheral foci of Albumin compared to the post 1 Gy TBI image (Red in image).
FIG. 8 depicts the histopathology of murine tongue one hour post 2 Gy TBI, longitudinal section. Note progressive destructive changes in the epithelial layer with disruption of the cornified spicule layer, increased scattered chromatin debris and edema of the sub-basement membrane layer.
FIG. 9 represents a MALDI image of murine tongue one hour post 3 Gy TBI, longitudinal section. Note loss of peripheral foci of albumin consistant with destructive changes in the epithelial layer and increase in central foci of Albumin compared to the post 2 Gy image.
FIG. 10 depicts the histopathology of murine tongue one hour post 3 Gy TBI, longitudinal section. Note progressive destructive changes and virtual complete loss of the cornified spicule layer corresponding to the loss of peripheral albumin foci (peripheral zone) in the MALDI image, increased scattered chromatin debris, and increased edema of the sub-basement membrane layer.
FIG. 11 represents the LAP monomer, light blue in the image.
FIG. 12 represents the LAP monomer, light blue and red in the image.
FIG. 13 represents the LAP monomer, light blue and red in the image.
FIG. 14 represents the Da Hemoglobin Subunit a, and Parathymosinare only found one hour post 1 Gy IR, red in the image.
FIG. 15 depicts a fatty acid-binding protein adipocyte seen 1 hour post 1 and 2 Gy IR, red in the image.
FIG. 16 demonstrates one hour post 2 Gy. Triosphosphate isomerase and superoxide dismutase are seen one hour post 2 Gy, but not seen in normal tissue or at one hour post 1 Gy. Red in the image.
FIG. 17 demonstrates hemaglobin α chains and BC011074 NID seen one hour post 3 Gy.



DETAILED DESCRIPTION OF THE INVENTION
As used herein, the following terms mean as follows:
As used herein, “radiation associated marker” means any molecule, such as a protein, peptide or fragment thereof whose presence, absence or amount in absolute quantity or in quantity relative to other molecules may be used as evidence of exposure to elevated levels of ionizing radiation. Stated differently, a radiation associated marker may be any molecule that may be used as a statistically significant predictor of exposure to elevated levels of ionizing radiation, or that may used as a statistically significant predictor of the amount of ionizing radiation to which an individual has been exposed, or that may be used as a statistically significant predictor or clinical outcome post exposure to elevated levels of ionizing radiation.
As used herein, a “predetermined value” is a standardized value based on a control. For example, a predetermined value can be based on an amount of radiation associated marker that is present in a biological sample obtained from an individual.
The term “amount” is used within the context of the analytical method used to measure the different radiation associated markers and may reflect a number, a concentration, etc., depending upon the analytical method chosen to measure the radiation associated markers.
The term “biological sample,” as used herein, generally refers to urine, saliva, serum, plasma, tears, or amniotic fluid. Buccal mucosa is an especially useful “biological sample” for the present invention because of its known sensitivity to ionizing radiation and easy accessibility for study.
The term “detecting” as used herein refers to identifying the presence of, identifying the presence of in relative amounts relative to another molecule or radiation associated marker relative to a predetermined value, or quantifying in absolute amounts.
The term “assessing exposure to ionizing radiation” includes quantifying the amount of ionizing radiation to which an individual has been exposed, quantifying the length of time to which an individual has been exposed to ionizing radiation, estimating or quantifying the amount of damage to a tissue or to an individual after exposure to ionizing radiation, and providing a prognosis of likely clinical sequelae or survival probability.
The term “elevated levels of ionizing radiation” encompasses instances where the ionizing radiation is present in amounts that are 10%, 20%, 30%, 50%, 75% or more greater than the amount of ionizing radiation experienced normally from environmental sources. The term “elevated levels of ionizing radiation” also encompasses instances where the ionizing radiation is present in amounts that are 2×, 3×, 4×, 5×, 10×, 25×, 50×, 100×, 500×, 1,000×, 5,000×, 10,000× 100,000×, 1,000,000× or more greater than the amount of ionizing radiation experienced normally from environmental sources. Ionizing radiation may be quantified and expressed in terms of a gray (Gy). One gray is the absorption of one joule of energy, in the form of ionizing radiation, by one kilogram of matter. For X-rays and gamma rays, these are the same units as the sievert (Sv). To avoid any risk of confusion between the absorbed dose and the equivalent dose, one must use the corresponding special units, namely the gray instead of the joule per kilogram for absorbed dose and the sievert instead of the joule per kilogram for the dose equivalent. The gray measures the deposited energy of radiation. The biological effects vary by the type and energy of the radiation and the organism and tissues involved. The sievert attempts to account for these variations. A whole-body exposure to 5 or more grays of high-energy radiation at one time usually leads to death within 14 days. This dosage represents 375 joules for a 75 kg adult (equivalent to the chemical energy in 20 mg of sugar). Since grays are such large amounts of radiation, medical use of radiation is typically measured in milligrays (mGy). The average radiation dose from an abdominal x-ray is 1.4 mGy, that from an abdominal CT scan is 8.0 mGy, that from a pelvic CT scan is 25 mGy, and that from a selective spiral CT scan of the abdomen and the pelvis is 30 mGy. One gray is equivalent to 100 rad. Therefore, the term “elevated levels of ionizing radiation” may be defined in some instances to mean a radiation dose greater than 0.25, 0.50, 1.0, 1.5, 2.0, 5.0, 10.0, 20.0, 50.0 or 100 mGy. In other instances, the term “elevated levels of ionizing radiation” may be defined to mean a radiation dose greater than 0.25, 0.50, 1.0, 1.5, 2.0, or 5.0 Gy.
According to an embodiment of this invention, the sample may be a saliva sample or a buccal swab. The sample may be obtained, for instance, about 15 minutes, 30 minutes, 1 hour, 2 hours, 6 hours, 12 hours, 1 day, 2 days or a week after exposure to elevated levels of ionizing radiation. In a particular embodiment of this invention, the sample is a buccal swab.
In addition, the present invention provides a method for determining the amount of radiation associated marker of in a sample comprising: (a) contacting the sample with an antibody which specifically binds to a radiation associated marker under conditions permitting formation of a complex between the antibody and the radiation associated marker; and (b) determining the amount of complexes formed thereby determining the amount of radiation associated marker in the sample.
This invention is illustrated in the experimental details section which follows. These sections are set forth to aid in an understanding of the invention but are not intended to, and should not be construed to limit in any way the invention as set forth in the claims which follow thereafter.
Radiation associated markers can be directly measured, for example, using anti-marker antibodies in an immunoassay, such as a Western blot or ELISA. Radiation associated markers can be indirectly measured, for example, using a capture antibody that binds the radiation associated marker.
The amount of radiation associated markers in a biological sample can be determined using any method known in the art, including, but not limited to, immunoassays using antibodies specific for the radiation associated marker. Any assay that functions to qualitatively or quantitatively determine variations in sample concentrations of radiation associated markers from normal levels can be employed in the practice of the invention.
For example, a monoclonal anti-radiation associated marker antibody can be generated by immunizing a mouse with the radiation associated marker. Once an immune response is detected, e.g., antibodies specific for the radiation associated marker are detected in the mouse serum, the mouse spleen is harvested and splenocytes are isolated. The splenocytes are then fused by well-known techniques to any suitable myeloma cells, for example, cells from cell line SP20 available from the American Type Culture Collection (ATCC). Hybridomas are selected and cloned by limited dilution. The hybridoma clones are then assayed by methods known in the art for cells that secrete antibodies capable of binding the radiation associated marker. Ascites fluid, which generally contains high levels of antibodies, can be generated by immunizing mice with positive hybridoma clones.
Any type of fusion phage, monoclonal, or polyclonal antibodies can be used in immunoassays of the invention, so long as the antibodies can be used in a reproducible fashion as markers for various radiation associated markers or as measures of the different levels of radiation associated markers observed in normal and variant populations.
In one embodiment, an amount of radiation associated marker can be measured using a capture antibody followed by a labeled secondary antibody using a strategy as described; for example, in U.S. Pat. No. 6,429,018, hereby incorporated by reference. The label on the secondary antibody can comprise any chemical, radioactive, lanthanide, colored dye, or genetic tag used in enzyme-linked immunosorbent assays (ELISAs), Western blots, and other sensitive and specific immunoassays and immunoradiometric assays using known methodology. These include conjugating the antibody with horseradish peroxidase or alkaline phosphatase that are easily measurable, typically using colorimetric, fluorometric or luminescent substrates. Genetic labels include firefly luciferase, employed because luciferase produces a bioluminescent molecule when incubated with its substrate, luciferin.
Serum albumin, by test strip, and numerous other novel protein and peptide biomarkers of ionizing radiation (IR) are consistently found by mass spectrometry in ex vivo wiping and histology sections of murine tongue epithelium one hour post 1, 2, and 3 Gy. Albumin and other proteins are not seen in non-irradiated tissue. Keratin is a ubiquitous component of tongue tissue, and can be seen at all levels of IR. The additional proteins in nonirradiated tissue, 1, 2, and 3 Gy are shown in the LCMS mass spectrometry Mascot search engine results. These biomarkers can be identified at POC with a simple oral cavity swab. FIG. 1. These biomarkers can be confirmed hours to days after the event with the same mass spectrometry techniques used in the initial biomarker identification with similar samples.
Matrix-Assisted Laser Desorption/Ionization (MALDI).
Matrix-assisted laser desorption/ionization (MALDI) is a soft ionization technique used in mass spectrometry, allowing the analysis of biomolecules (biopolymers such as proteins, peptides and sugars) and large organic molecules (such as polymers, dendrimers and other macromolecules), which tend to be fragile and fragment when ionized by more conventional ionization methods. It is most similar in character to electrospray ionization both in relative softness and the ions produced (although it causes many fewer multiply charged ions). The ionization is triggered by a laser beam (normally a nitrogen laser). A matrix is used to protect the biomolecule from being destroyed by direct laser beam and to facilitate vaporization and ionization.
The matrix consists of crystallized molecules, of which the three most commonly used are 3,5-dimethoxy-4-hydroxycinnamic acid (sinapinic acid), α-cyano-4-hydroxycinnamic acid (alpha-cyano or alpha-matrix) and 2,5-dihydroxybenzoic acid (DHB). A solution of one of these molecules is made, often in a mixture of highly purified water and an organic solvent (normally acetonitrile (ACN) or ethanol). Trifluoroacetic acid (TFA) may also be added. A good example of a matrix-solution would be 20 mg/mL sinapinic acid in ACN:water:TFA (50:50:0.1). The identity of suitable matrix compounds is determined to some extent by trial and error, but they are based on some specific molecular design considerations: they have a low molecular weight; they are acidic and act as a proton source to encourage ionization of the analyte; they have a strong optical absorption in the UV and efficiently absorb the laser irradiation; and they are functionalized with polar groups allowing use in aqueous solutions.
The matrix solution is mixed with the analyte (e.g. protein sample). The organic solvent allows hydrophobic molecules to dissolve into the solution, while the water allows for water-soluble (hydrophilic) molecules to do the same. This solution is spotted onto a MALDI plate (usually a metal plate designed for this purpose). The solvents vaporize, leaving only the recrystallized matrix, but now with analyte molecules spread throughout the crystals. The matrix and the analyte are said to be co-crystallized in a MALDI spot.
The laser is fired at the crystals in the MALDI spot. The matrix absorbs the laser energy, and the matrix is ionized by this event. The matrix transfers part of its charge to the analyte molecules (e.g. protein) thereby ionizing them while still protecting them from the disruptive energy of the laser. Ions observed after this process consist of a neutral molecule [M] and an added or removed ion. Together, they form a quasimolecular ion, for example [M+H]+ in the case of an added proton, [M+Na]+ in the case of an added sodium ion, or [M−H]− in the case of a removed proton. MALDI is capable of creating singly-charged ions, but multiply charged ions ([M+nH]n+) can also be created, as a function of the matrix, the laser intensity and/or the voltage used. Note that these are all even-electron species. Ion signals of radical cations can be observed eg. in case of matrix molecules and other stable molecules.
Atmospheric pressure (AP) matrix-assisted laser desorption/ionization (MALDI) is an ionization technique (ion source) that in contrast to vacuum MALDI operates at normal atmospheric environment. In vacuum MALDI, ions are typically produced at 10 mTorr or less while in AP-MALDI ions are formed in atmospheric pressure.
AP-MALDI is used in mass spectrometry (MS) in a variety of applications including proteomics and drug discovery fields. AP-MALDI mass spectrometry is often used in proteomics, DNA/RNA/PNA, lipids, oligosaccharides, phosphopeptides, bacteria, small molecules and synthetic polymers, similar applications as available also for vacuum MALDI instruments.
The AP-MALDI ion source is easily coupled to an ion trap mass spectrometer or any other MS system equipped with ESI (electrospray ionization) or nanoESI source.
The type of a mass spectrometer most widely used with MALDI is the TOF (time-of-flight mass spectrometer) because of its large mass range. The TOF measurement procedure is suited to the MALDI ionization process since the pulsed laser takes individual ‘shots’ rather than working in continuous operation. MALDI-TOF instruments are typically equipped with an “ion mirror,” deflecting ions with an electric field thereby doubling the ion flight path and increasing the resolution. Today, commercial reflectron TOF instruments reach a resolving power m/Δm of well above 20′000 FWHM (full-width half-maximum, Δm defined as the peak width at 50% of peak height).
In proteomics, MALDI is used for identifying proteins isolated through gel electrophoresis: SDS-PAGE, size exclusion chromatography, and two-dimensional gel electrophoresis. One method used is peptide mass fingerprinting by MALDI-MS, or with post ionization decay or collision-induced dissociation.
IMS.
The history of IMS (Imaging Maldi Spectroscopy) began with single cell studies of Aplysia californica neurons by matrix-assisted laser desorption/ionization time-of flight mass spectrometry (MALDI/TOF-MS). This was probably the first direct tissue MALDI identification of peptides and tissue profiling based on ion density. (Garden et al., J Mass Spectrom 1996; 31:1126-30) A refinement of this technique, imaging MALDI was described in human buccal mucosa, and rat pituitary and pancreas glands using two different approaches: direct targeting of the tissue itself and by analysis of blotted targets previously exposed to the tissue. (Caprioli et al., Anal Chem 1997; 69:4751-60) An “MS Image Tool,” using the peptide neurotensin (peak at m/z 1674) significantly improved the data acquisition, speed of IMS, and utilization of the technique. (Stoeckli et al., J Am Soc Mass Spectrom 1999; 10:67-71) In a laser, capture microdissection (LCM) study of both tumor and normal human breast tissue fixed in ethyl alcohol, and stained with hematoxylin and eosin, normal tissue, carcinoma in situ, invasive carcinoma, and metastatic carcinoma could be distinguished by their different MALDI spectra. The introduction of “BioMap” software, by applying a baseline correction to the spectra and integrating over the peak of interest, demonstrated mouse brain images of amyloid 13, and A13 peptides. That report was proof of the principle that MALDI images of tissue could be obtained based upon the mass spectrometry mass/charge, m/z peak of interest. (Stoeckli et al., Nat Med 2001; 7:493-6) Subsequent profiling and IMS of normal mouse epididymis identified different protein activity (ion densities) throughout the sections. (Chaurand et al., Electrophoresis 2002; 23:3125-35)
The negative effects on IMS resolution from destructive tissue freezing artifacts, excessive dehydration due to ethanol fixation, paraformaldeyde cationization, embedding artifacts from OCT polymer and agar, and coarse matrix crystal size were first described in a report on spatial profiling of invertebrate ganglia. (Kruse et al., J Am Soc Mass Spectrom 2003; 14:752-9) In a follow-up report, they suggested a solution to the problem of freezing artifacts using glycerol and the related compounds ethane-1,2-diol and propane-1,2-diol to stabilize cellular membranes. (Rubakhin et al., Anal Chem 2003; 75:5374-80) A subsequent report suggested direct liquid nitrogen immersion of tissue in aluminum wrapping as a means of rapid fixation, but ignored the known consequences of freezer artifact. (Schwartz et al., J Mass Spectrom 2003; 38:699-708) Adjunctive histologic staining with methylene blue stained tissues on standard metal plates or indium-tin coated glass slides were shown to be compatible with IMS, but cresyl violet stain decreased IMS signal intensity. (Chaurand et al., Anal Chem 2004; 76:1145-55)
Tissue blotting with trypsin digestion for MSMS (mass spectrometry mass spectrometry using two mass spectrometers in tandem) data base analysis was shown to be useful in analyte localization, but was destructive to tissue morphology. (Bunch et al., Rapid Communications in Mass Spectrometry 2004; 18:3051-60) Others described a less destructive trypsin digest step to their prior tissue blotting technique for IMS. (Rohner et al., Mech Ageing Dev 2005; 126:177-85) Another method, matrix-enhanced secondary ion mass spectrometry (ME) SIMS was described and used for direct molecular imaging of the ganglia of the freshwater snail, Lymnaea stagnalis. (Altelaar et al., Anal Chem 2005; 77:735-41) However, this technique presented significant limitations and was proved unsuitable for direct tissue imaging. A refinement of IMS, oversampling with complete sample ablation at each sample position on the target plate, provided significant resolution enhancement with a translation stage raster step size of 25 μM. A 40 μM object could now be resolved with a 100 μM laser. (Jurchen et al., Journal of the American Society for Mass Spectrometry 2005; 16:1654-9)
Tissue treatments with organic solvents such as chloroform, acetone, hexane, toluene, or xylene were shown to be an effective and rapid method for signal enhancement in MALDI direct tissue profiling. (Lemaire et al., Analytical Chemistry 2006; 78:7145-53) These studies demonstrated that solvent treatments partially removed lipids from the tissue surface. Compared to previous studies with ethanol, chloroform/xylene solvent, rinsing is more specific for lipid removal and does not generate delocalization or extraction of most soluble peptides/proteins as tested by immuno-histochemistry experiments. Among all the tested solvents, chloroform and xylene produced the greatest increase in MALDI signal intensity and number of detected peptides/proteins. However, this treatment does not reduce salt adducts as does alcohol treatment. The results suggest that it is possible to detect, after organic rinsing treatments, compounds, such as peptides/proteins present in the cytoplasm, that were masked by lipids in the tissue.
Clench et al. reported the development and application of a method using 9-aminoacridine as a matrix for negatively charged ions in MALDI imaging. (Burrell et al., J Exp Bot 2007; 58:757-63) Crossman demonstrated the need for thin sections to avoid differential extraction efficiency of matrix solvent in different tissues. (Crossman et al., Rapid Communications in Mass Spectrometry 2006; 20:284-90) Pevsner demonstrated direct cellular MALDI identification of proteins in fixed cells and tissues without freezer artifact, tissue corrosion by matrix solvents or the use of tissue blotting. This was confirmed in a later report. (Pevsner et al., Direct identification of proteins from cells and tissues using MALDI TOF. Anal Chem.; Pevsner et al., J Soc Gynecol Investig 2006; 13:A1-B10; Groseclose et al., J Mass Spectrom 2007; 42:254-62)
Metal-assisted (MetA) secondary ion mass spectrometry (SIMS), a variation on SIMS as well as matrix-assisted laser desorption/ionization (MALDI) IMS can provide images from tissue, but the duration of these protocols were highly dependent on sample size and technique parameters. The duration of these studies averaged approximately 5 h. (Altelaar et al., Nat Protoc 2007; 2:1185-96)
Agar et al. studied multiple solvent/matrix combinations. However, the tissue sections at the electron microscopic level demonstrated both freezing artifact and structural distortion, indicating that their method disrupts normal subcellular structures such as mitochondria. (Agar et al., Matrix Solution Fixation: Histology-Compatible Tissue Preparation for MALDI Mass Spectrometry Imaging. Anal Chem 2007;.) Baluya et al reported a variation on inkjet-printed matrix application to tissue specimens previously described by Sloane et al. (Baluya et al., Anal Chem 2007; 79:6862-7; Sloane et al., Mol Cell Proteomics 2002; 1:490-9) The matrix application was of better quality and more reproducible than from specimens prepared by the electrospray and airbrush methods, but still was not completely uniform. A uniform method of tissue matrix application is sublimation. Sublimation is solvent free, rapid, and was successfully used to identify lipids in brain tissue, and more recently described for proteins or peptides. The challenge of IMS in formalin fixed paraffin embedded tissue was first addressed by and then by Pevsner and later by Stauber. (Hankin et al., J Am Soc Mass Spectrom 2007; 18:1646-52; Pevsner et al. “Microtubule Associated Proteins (MAP) and Motor Molecules: Direct Tissue MALDI Identification and Imaging.” 2007; Pevsner et al., “Colon Cancer: Protein Biomarkers in Tissue and Body.” 2007; Pevsner et al., “Colorectal Carcinoma—Field Defects in SatelliteTissue.” 2007; Puolitaival et al., J Am Soc Mass Spectrom 2008; 19:882-6; Lemaire et al., J Proteome Res 2007; 6:1295-305; 48. Stauber et al., J Proteome Res 2008; 7:969-78)
This invention is illustrated in the experimental section that follows. These examples are set forth to aid in understanding the invention but are not intended to, and should not be construed to, limit in any way the invention as set forth in the claims.
EXAMPLE 1
New protein biomarkers were identified in murine buccal mucosa after ionizing radiation. The new protein biomarkers were identified from scraping of buccal mucosa obtained fifteen and thirty minutes post ionizing radiation exposure. The biomarkers are as follows:

    
    
        DnaJ homolog subfamily C member 1 (DnaJ protein homolog MTJ1) gi|2494160|sp|Q61712.1|DNJC1_MOUSE[2494160. DnaJ domain. DnaJ domains (J-domains) are associated with hsp70 heat-shock system and it is thought that this domain mediates the interaction which is triggered by ionizing radiation.
        mCG1463, isoform CRA_b [Mus musculus]
        gi|148683958|gb|EDL15905.1|[14868395]. This protein is involved in transcriptional regulation which is triggered by ionizing radiation.
    
    


The following three proteins all represent biomarkers of acute radiation exposure which triggers either transcription or post-translational modification.

    
    
        Novel protein (B230312A22Rik) [Mus musculus] 955.87 Da
        Amino Acid Sequence: MSGPRKAPED
        gi:45593111 Demonstrated 15 minutes post ionizing radiation.
        Unnamed protein product [Mus musculus] 1184.12 Da
        Amino Acid Sequence: AKDYYGTGYF
        gi|1333908| Demonstrated 15 minutes post ionizing radiation.
        Transaldolase [Mus musculus] 939.07 Da
        Amino Acid Sequence: MRHVQAELS
        gi|168984609| Demonstrated 30 minutes post ionizing radiation.
    
    


The identification of specific protein biomarker profiles and new protein biomarkers of ionizing radiation exposure enable reliable associations with dose-exposure, for biodosimetry purposes. This strategy will result in the development of diagnostic tests using a buccal mucosa swab to provide acute clinical quantitative evidence of radiation exposure of 3 Gy or more and allow for accurate and immediate triage of victims of an ionizing radiation mass casualty.
EXAMPLE 2
Fourteen swiss mice were anesthetized with intraperitoneal ketamine-xylazine (k/x, 80/10 mg/kg ip). Ten mice received 3 Gy to the head. Buccal mucosa scrapings were obtained fifteen minutes (5 mice) and thirty minutes (five mice) post exposure. Buccal mucosa scrapings from the four non-radiated mice were used as controls. All reagents were obtained from Sigma (St. Louis, Mo.) and used unmodified. All samples were placed in 1 mL of 100 mMolar ammonium bicarbonate buffer. Proteins were extracted from all the tissues in this buffer with high pressure (Barocycler, Pressure BioSciences, South Easton, Mass.). The samples were concentrated by lyopholization to 200 μL and divided into three components. One for trypsin digestion and liquid chromatography mass spectrometry, LCMS, analysis (bottoms-up proteomics), one for HPLC protein separation and FTMS analysis (top-down proteomics) to identify post-translational modifications (this aliquot was frozen for later analysis), and one for direct identification with matrix assisted laser desorption mass spectrometry, MALDI analysis. The study was conducted under an approved protocol of the New York University School of Medicine Institutional Animal Care and Use Committee, Laboratory Animal Protocol #080601-01.
Sinapic acid (5 mg/mL) 0.3 μL was pipetted onto a conductive MALDI plate, allowed to dry, and covered with an equal amount of sample. All fourteen samples were examined with MALDI in linear mode. The findings were consistent across all four controls, 15 minute (N=5), and 30 minute (N=5) samples, FIG. 3. Cyano 4 hydroxy cinnamic acid (5 mg/mL) 0.3 μL was pipetted onto a conductive MALDI plate, allowed to dry, and covered with an equal amount of sample. All fourteen samples were examined with MALDI in reflectron mode. The findings were consistent across all four controls, 15 minute (N=5), and 30 minute (N=5) samples, FIG. 4.
Results.
The linear mode MALDI MS experiment is shown in FIG. 3. The lowest spectrum is a control sample. The middle spectrum is a 15 minute post 3 Gy sample. The upper spectrum is a 30 minute post 3 Gy sample. At 15 minutes post 3 Gy, a protein 63870.32 Da, D naJ homolog subfamily C member 1 (DnaJ protein homolog MTJ1) gi|2494160|sp|Q61712.1|DNJC1_MOUSE[2494160] was demonstrated. DnaJ domains (J-domains) are associated with hsp70 heat-shock system and it is thought that this domain mediates the interaction. At 30 minutes post 3 Gy, a protein 72449.25 Da, mCG 1463, isoform CRA_b [Mus musculus] gi|148683958|gb|EDL15905.1|[14868395] is tentatively identified in the spectrum. This protein is involved in transcriptional regulation. At 15 and 30 minutes post 3 Gy, the spectrum of peptides is demonstrated in FIG. 4, and the peptide amino acid sequences derived with bioinformatics and the corresponding proteins they represent are shown in Table 1.







TABLE 1





Peptide amino acide sequence and corresponding proteins 


identified with bioinformatics at 15 and 30 minutes post 3 Gy.


Peptides at 15 and 30 min post 3 Gy



















15 min 
955.87
gi|4559311| novel protein(B230312A22Rik)
msgprkaped
(SEQ ID NO: 1) 




[Mus musculus]





1184.12
gi|1333908|unnamed protein product
akdyygtgyf
(SEQ ID NO: 2)




[Mus musculus]




30 min
939.07
gi|168984609|transaldolase [Mus musculus].
mrhvqaels
(SEQ ID NO: 3)









These experiments demonstrated novel changes 15 and 30 minutes post ionizing radiation to the head and neck.
EXAMPLE 3
Additional experiments using total body ionizing radiation (N=21), TBI, exposure at 0 (N=3), 1 (N=6), 2 (N=6), and 3 (N=6) Gy were performed (mice). The murine tongue was chosen for examination. The mice were sacrificed at one hour post TBI, MALDI imaging of tongue tissue was combined with high resolution, 1.43 nm, (IR filter visible light 400 nm, Zeiss 63X oil immersion 1.4 NA objective, and 1.4 NA Olympus condenser) bright field microscopy (the smallest mitochondria are 1000 nm); and liquid chromatography mass spectrometry, LCMS, to obtain protein sequence data for exact identification and localization of post TBI protein biomarkers in the tissue sections. Protein extraction from the 3rd contiguous section was performed with a Barocycler and digested with trypsin for LCMS examination. Contiguous 5μ cryo-sections were obtained for MALDI imaging, examples of normal, post 1, 2, and 3 Gy, FIGS. 5-10; histology (hematoxylin and eosin, H&E), examples of normal, post 1, 2, and 3 Gy, FIGS. 5-10; and nanoflow liquid chromatography mass spectrometry, LCMS, examples of normal, post 1, 2, and 3 Gy. The molecular weights of the identified proteins were used in the MALDI image prepared with sublimation for tissue localization. (Naftolin et al., Reproductive Sciences 2007; 14:257A; Pevsner et al., Biomarkers Med 2009; 3:55. FIG. 5 represents a MALDI image of murine tongue one hour post 1 Gy TBI, longitudinal section. Note peripheral foci of Albumin (Red in image). FIG. 6 demonstrates the histopathology of murine tongue one hour post 1 Gy TBI, longitudinal section. Note minimal destructive changes in the epithelial cornified spicule layer and edema in the basal region. FIG. 7 represents a MALDI image Note marked increase in peripheral foci of Albumin compared to the post 1 Gy TBI image (Red in image).
FIG. 8 demonstrates the histopathology of murine tongue one hour post 2 Gy TBI, longitudinal section. Note progressive destructive changes in the epithelial layer with disruption of the cornified spicule layer, increased scattered chromatin debris and edema of the sub-basement membrane layer. FIG. 9 represents a MALDI image of murine tongue one hour post 3 Gy TBI, longitudinal section. Note loss of peripheral foci of albumin consistant with destructive changes in the epithelial layer and increase in central foci of Albumin compared to the post 2 Gy image. FIG. 10 demonstrates the histopathology of murine tongue one hour post 3 Gy TBI, longitudinal section. Note progressive destructive changes and virtual complete loss of the cornified spicule layer corresponding to the loss of peripheral albumin foci (peripheral zone) in the MALDI image, increased scattered chromatin debris, and increased edema of the sub-basement membrane layer.







TABLE 2





{MATRIX SCIENCE} Mascot Search Results
















User:
NYUSOM


Email:
seagea01@nyumc.org


Search title:



MS data file:
E:\20090324_LCMS_Samples\1_001_CID.mgf


Database:
MSDB 20060831 (3239079 sequences; 1079594700 residues)


Taxonomy:
Mus musculus (house mouse) (90914 sequences)


Timestamp:
Apr. 8, 2009 at 13:25:26 GMT







Protein hits:








AAH11074 
BC011074 NID: - Mus musculus


K1C17_MOUSE
Keratin, type I cytoskeletal 17 (Cytokeratin-17) (CK-17) (Keratin-17) (K17). - 



Mus musculus (Mouse).


Q2M1G8_MOUSE
2410039E07Rik protein (Fragment). - Mus musculus (Mouse).


KRMSE1
keratin, 59K type I cytoskeletal - mouse


K1C15_MOUSE
Keratin, type I cytoskeletal 15 (Cytokeratin-15) (CK-15) (Keratin-15) (K15). -



Mus musculus (Mouse).


Q32P04_MOUSE
Krt2-5 protein (Fragment). - Mus musculus (Mouse).


Q8BGZ7_MOUSE
10 days neonate skin cDNA, RIKEN full-length enriched library, clone: 4732475I03



product: CYTOKERATIN homolog (10 days neonate skin CDNA, RIKEN full-length



enriched library, clone: 4732468K03 product: CYTOKERATIN TYPE II homolog) (6 days



neonate skin cD


K2C6A_MOUSE
Keratin, type II cytoskeletal 6A (Cytokeratin-6A) (CK 6A) (K6a keratin) (Keratin-



ε alpha) (mK6-alpha). - Mus musculus (Mouse).


A55682
keratin 13, type I cytoskeletal - mouse


Q8K2E4_MOUSE
CDNA sequence BC031593. - Mus musculus (Mouse).


JQ0028
cytokeratin 19 - mouse


Q9D2K8_MOUSE
0 day neonate head cDNA, RIKEN full-length enriched library, clone: 4833436C19



product: keratin complex 2, basic, gene 1, full insert sequence. - Mus musculus



(Mouse).


KRMS2
keratin, type II cytoskeletal - mouse (fragment)


Q3TTY5_MOUSE
10 days neonate skin cDNA, RIKEN full-length enriched library, clone: 4732404G19



product: keratin complex 2, basic, gene 17, full insert sequence (10 days neonate



skin cDNA, RIKEN full-length enriched library, clone: 4732426A12 product: keratin



complex


S07330
keratin, epidermal - mouse


A26135
keratin, 52K type I epidermal (clone pke 52) - mouse (fragment)


Q80VP7_MOUSE
Hypothetical protein MGC54654. - Mus musculus (Mouse).


Q6IFT3_MOUSE
Keratin Kb40. - Mus musculus (Mouse).


Q8BIS2_MOUSE
10 days neonate skin cDNA, RIKEN full-length enriched library, clone: 4732456N10



product: similar to KERATIN, TYPE II CYTOSKELETAL 6D (CYTOKERATIN 6D) (CK 6D) (K6D



KERATIN). - Mus musculus (Mouse).


Q6NXH9_MOUSE
Type II keratin Kb36. - Mus musculus (Mouse).


K2C1B_MOUSE
Keratin, type II cytoskeletal 1b (Type II keratin Kb39) (Embryonic type II



keratin-1). - Mus musculus (Mouse).


BAE40567
AK168726 NID: - Mus musculus


K2C4_MOUSE
Keratin, type II cytoskeletal 4 (Cytokeratin-4) (CK-4) (Keratin-4) (K4)



(Cytoskeletal 57 kDa keratin). - Mus musculus (Mouse).


Q3UV17_MOUSE
Adult female vagina cDNA, RIKEN full-length enriched library, clone: 9930024P18



product: similar to Keratin 2p. - Mus musculus (Mouse).


I59009
epidermal keratin subunit II - mouse


Q9Z1R9_MOUSE
Trypsinogen 16 (Protease, serine, 1). - Mus musculus (Mouse).


Q9CXH5_MOUSE
17 days embryo head cDNA, RIKEN full-length enriched library, clone: 3300001P16



product: hemoglobin, beta adult major chain, full insert sequence. - Mus musculus



(Mouse).


Q6IME9_MOUSE
Type-II keratin Kb35. - Mus musculus (Mouse).


Q9JJ20_MOUSE
14-3-3 protein sigma - Mus musculus (Mouse).


H2B1B_MOUSE
Histone H2B type 1-B (h2B-143). - Mus musculus (Mouse).


Q8BKC6_MOUSE
0 day neonate eyeball cDNA, RIKEN full-length enriched library, clone: E130120J09



product: hypothetical Intermediate filament proteins containing protein, full



insert sequence. (Fragment). - Mus musculus (Mouse).


I48739
MHa2 (keratin acidic 2) - mouse


H2A2A_MOUSE
Histone H2A type 2-A (H2A.2). - Homo sapiens (Human).


HAMS
hemoglobin alpha chains - mouse


BAC33789
AK049517 NID: - Mus musculus





Probability Based Mowse Score


Ions score is −10 * Log (P), where P is the probability that the observed match is a random event.


Individual ions scores > 38 indicate identity or extensive homology (p < 0.05).


Protein scores are derived from ions scores as a non-probabilistic basis for ranking protein hits.






Table 2 provides the results of a Mascot search of proteins in normal murine tongue. No albumin was identified.







TABLE 3





Peptide Summary Report(../data/20090408/FtiAifuwe.dat)


{MATRIX SCIENCE} Mascot Search Results
















User
NYUSOM


Email
seagea01@nyumc.org


Search title



MS data file
E:\20090324_LCMS_Samples\9_001_CID.mgf


Database
MSDB 20060831 (3239079 sequences; 1079594700 residues)


Taxonomy
Mus musculus (house mouse) (90914 sequences)


Timestamp
8 Apr. 2009 at 13:57:09 GMT









Protein hits
Q8BGZ7_MOUSE
10 days neonate skin cDNA, RIKEN full-length enriched library, clone: 4732475I03




product:CYTOKERATIN homolog (10 days neonate skin cDNA, RIKEN full-length




enriched library, clone: 4732468K03 product: CYTOKERATIN TYPE (1 homolog) 




(6 days neonate skin cD



K2C1-MOUSE
Keratin, type I: cytoskeletal 1 (Cytokeratin-1) (CK-1) (Keratin-1) (K1) (67 kDa




cytokeratin).- Mus musculus (Mouse).



Q9D2K8_MOUSE
0 day neonate head cDNA, RIKEN full-length enriched library, clone: 4833436C19




product: keratin complex 2, basic, gene 1, full insert sequence.- Mus musculus




(Mouse).



Q32P04_MOUSE
Krt2-5 protein (Fragment).- Mus musculus (Mouse).



K2C6A_MOUSE
Keratin, type II cytoskeletal 6A (Cytokeratin-6A; (CV 6A) (K6a keratin) (Keratin-




6 alpha) (mk6-alpha).- Mus musculus (Mouse).



Q80VPP7_MOUSE
Hypothetical protein MGC54654.- Mus musculus (Mouse).



Q8BIS2_MOUSE
10 days neonate skin cDNA, RIVEN full-length enriched library, clone: 4732456N10




product: similar to KERATIN, TYPE II CYTOSKELETAL 6D (CYTOKERATIN 6D) 




(CK CD) (K6D KERATIN).- Mus musculus (Mouse).



Q9CXH5_MOUSE
17 days embryo head cDNA, RIKEN full-length enriched library, clone: 3300001P16




product: hemoglobin, beta adult major chain, full insert sequence.- Mus musculus




(Mouse)



KRMSE1
keratin, 59K type I cytoskeletal - mouse



Q6NXH9_MOUSE
Type II keratin in Kb36.- Mus musculus (Mouse).



K2C1B_MOUSE
Keratin, type II cytoskeletal lb (Type II keratin Kb39) (Embryonic type II




keratin-1).- Mus musculus (Mouse).



Q6IFZ9_MOUSE
TYPE II keratin Kb37.- Mus musculus (Mouse).




10 days neonate skin cDNA, RIKEN full-length enriched library, clone: 4732404G19




product: keratin complex 2, basic, gene 17, full insert sequence (10 days neonate




skin cDNA, RIKEN full-length enriched library, clone: 4732426A12 product: keretin




complex



Q6IFT3_MOUSE
Keratin, Kb40.- Mus musculus (Mouse).



Q3UV17_MOUSE
Adult female vagina cDNA, RIKEN full-length enriched library, clone: 9930021P18




product:similar to Keratin 2p.- Mus musculus (Mouse).









Table 3 lists LCMS identified proteins with Mascot search of proteins in murine tongue one hour post 1 Gy TBI. Albumin appears at the end of the list and corresponds to the findings in the MALDI image one hour post 1 Gy.







TABLE 4





Peptide Summary Report(../data/20090408/FtiAifsSE.dat)


{MATRIX SCIENCE} Mascot Search Results
















User
NYUSOM


Email
seagea01@nyumc.org


Search title



MS data file
E:\20090324_LCMS_Samples\10_001_CID.mgf


Database
MSDB 20060831 (3239079 sequences; 1079594700 residues)


Taxonomy
Mus musculus (house mouse) (90914 sequences)


Timestamp
8 Apr. 2009 at 14:03:15 GMT









Protein hits
KRMSE1
keratin, 59K type I cytoskeletal - mouse



Q9CY54_MOUSE
13 days embryo liver cDNA, RIKEN full-length enriched library, 




clone: 2500004H04 product: hemoglobin, beta adult major chain, 




full insert sequence.- Mus musculus (Mouse).



HAMS
hemoglobin alpha grains - mouse



Q9CY06_MOUSE
13 days embryo liver cDNA, RIKEN full-length enriched library, clone: 2510040P05




product: hemoglobin, beta adult major chain, full insert sequence.- Mus musculus




(Mouse).



AAH11074
BC011074 NID: - Mus musculus 



HBMS
hemoglobin beta major chain - mouse



Q3TTY5_MOUSE
10 days neonate skin cDNA, RIKEN full-length enriched library, 




clone: 4732404G19 product:keratin complex 2, basic, gene 17, full insert sequence 




(10 days neonate skin cDNA, RIKEN full-length enriched library, 




clone: 4732426A12 product:keratin complex



Q3UJH8_MOUSE
16 days embryo heart cDNA, RIKEN full-length enriched library, clone: I32000IF12




product: glutamate oxaloacetate transarunaso 1, soluble, full insert sequence.-




Mus musculus(Mouse)



Q8BGZ7_MOUSE
10 days nenoate skin cDNA, RIKEN full-length enriched library, clone: 4732475I03




product:CYTOKERATIN homolog (10 days neonate skin cDNA, RIKEN 




full-length enriched library, clone: 4732468K03 product: CYTOKERATIN TYPE II 




homolog) (6 days neonate skin cD



K1C17_MOUSE
Keratin, type I cytoskeletal 17 (Cytokeratin-17) (CK-17) (Keratine-17) (K17).-




Mus musculus (Mouse).



Q9R0S6_MOUSE
Beta-1-globin (Fragment).- Mus musculus (Mouse).



K2C1_MOUSE
Keratin, type II cytoskeletal 1 (Cytokeratin-1) (CK-1) (Keratin-1) (K1) (67 kDa)




cytokeratin).- Mus musculus (Mouse).



Q9D2K8_MOUSE
0 day neonate head cDNA, RIKEN full-length enriched library, clone: 4833436C19




product: keratin complex 2, basic, gene 1, full insert sequence.- Mus musculus




(Mouse).



A55682
keratin 13, type I cytoskeletal - mouse









Table 4 lists LCMS identified proteins with Mascot search of proteins in murine tongue one hour post 2 Gy TBI. Albumin appears next to last in the list.







TABLE 5





Peptide Summary Report(../data/20090408/FtiAifsEL.dat)


{MATRIX SCIENCE} Mascot Search Results
















User
NYUSOM


Email
seagea01@nyumc.org


Search title



MS data file
E:\20090324_LCMS_Samples\16_001_CID.mgf


Database
MSDB 20060831 (3239079 sequences; 1079594700 residues)


Taxonomy
Mus musculus (house mouse) (90914 sequences)


Timestamp
8 Apr. 2009 at 14:05:43 GMT









Protein hits
HAMS
hemoglobin alpha chains - mouse



Q9CY12_MOUSE
13 days embryo liver cDNA, RIKEN full-length enriched library, 




clone: 2510039D09 product: hemoglobin, beta adult major chain, 




full insert sequence.- Mus musculus (Mouse).



Q8BGZ7_MOUSE
10 days neonate skin cDNA, RIKEN full-length enriched library, 




clone: 4732475I03 product: CYTOKERATIN homolog (10 days neonate skin 




cDNA, RIKEN full length enriched library, clone: 4732468K03 




product: CYTOKERATIN TYPE II homolog) (6 days neonate skin cD



Q32P04_MOUSE
Krt2-5 protein (Fragment).- Mus musculus (Mouse).



Q3TV03_MOUSE
Adult male stomach cDNA, RIKEN full-length enriched library, 




clone: 2210414C06 product:albumin 1, full insert sequence.- Mus musculus




(Mouse).



KRMSE1
keratin, 59K type I cytoskeletal - mouse



K1C15_MOUSE
Keratin, type I cytoskeletal 15 (Cytokeratin-15) (CK-15) (Keratin-15) (K15).-




Mus musculus (Mouse).



Q8BIS2_MOUSE
10 days nenoate skin cDNA, RIKEN full-length enriched library, 




clone: 4732456N10 product:smilar to KERATIN, TYPE II CYTOSKELETAL 




6D (CYTOKERATIN 6D) (CK 6D) (KcD KERATIN).- Mus musculus (Mouse).





Probability Based Mowse Score


Ions score is −10 * Log(P), where P is the probability that the observed match is a random event.


Individual ions scores > 38 indicate identity or extensive homology (p < 0.05).


Protein scores are derived from ions scores as a non-probabilistic basis for ranking protein hits.






Table 5 lists LCMS identified proteins from two separate samples 16 and 19. Mascot search of proteins in murine tongue one hour post 3 Gy TBI. The absence of albumin in these two 3 Gy samples, it was present in other 3 Gy samples (data not shown) is consistent with the marked tissue destruction, and very small amount noted in the MALDI image.
EXAMPLE 4
It has been shown (MHB) that latent transforming growth factor-β, LTGF-β, is rapidly activated in vivo after ionizing radiation (IR). This is evidenced by increased immunoreactivity of TGFβ epitopes that are masked by the latent complex. (Barcellos-Hoff Mol Biol Cell 1994; 5:139a; Barcellos-Hoff et al., Am J Pathol 1995; 147:1228-37; Barcellos-Hoff et al., Molec Endocrin 1996; 10:1077-83; Barcellos-Hoff et al., Mol Endocrinol 1996; 10:1077-83; Barcellos-Hoff Mammary Gland Biol Neoplasia 1996; 1:353-63; Ehrhart et al., FASEB J 1997; 11:991-1002; Barcellos-Hoff et al., Breast Cancer Res 2000; 2:92-9; Barcellos-Hoff et al., Nat Rev Cancer 2005; 5:867-75; Jobling et al., Radial Res 2006; 166:839-48; Jobling et al., Radial Res 2006; 166:839-848). Because reactive oxygen species, ROS, are a product of the interaction of IR with water or with cell membranes, it was postulated that the rapid activation of TGFβ in vivo could be due to ROS generated by IR if the protein itself contained redox sensitive amino acids. We were able to demonstrate the monomers of TGFβ and the latency associated protein, LAP, in MALDI images one hour post 1, 2, and 3 Gy, FIGS. 18,19, 20. Note the approximately 10 Da variance in all three images. This molecular weight variance and spatial resolution can be improved, infra vida. Therefore TGFβ can also be used as a protein biomarker of absorbed dose. Quantization can be obtained from trypsin digests studied by LCMS. FIGS. 11, 12 and 13 depict LAP monomers, light blue in the image.
Additional dose specific protein biomarkers identified from other LCMS studies were not seen in normal tissue. Hemoglobin subunit α, and parathymosinare only found one hour post 1 Gy; Fatty Acid Binding Protein Adipocyte seen 1 hour post 1 and 2 Gy IR; and triosphosphate Isomerase, and superoxide dismutase seen at one hour post 2 Gy are not seen in normal tissue or at one hour post 1 Gy.
FIG. 14 depicts da hemoglobin subunit α, and parathymosinare only found one hour post 1 Gy IR, red in the image. FIG. 15 depicts fatty acid-binding protein adipocyte seen 1 hour post 1 and 2 Gy IR, red in the image. FIG. 16 depicts one hour post 2 Gy exposure. Triosphosphate isomerase and superoxide dismutase are seen one hour post 2 Gy, but not seen in normal tissue or at one hour post 1 Gy (red in the image). FIG. 17 demonstrates hemaglobin α chains and BC011074 NID seen one hour post 3 Gy exposure.
The results with MALDI imaging, high resolution microscopy, and LCMS are proof of principle that post TBI protein biomarkers can be identified and used as measures of absorbed radiation dose at 1, 2, and 3 Gy with our current technology. Identification of a larger library of post TBI proteins will improve specificity of both the POC triage kit and the HT device. This will dramatically limit false positives and false negatives. This will be achieved with a combination of strategies including higher resolution bright field microscopy, atomic force microscopy of irradiated tissue for higher spatial resolution of tissue loci of proteins by co-registration with the MALDESI images. (Collier et al., Anal Chem 2008; 80:4994-5001; Bereman et al., Rapid Commun Mass Spectrom 2008; 22:1563-6; Sampson et al., J Am Soc Mass Spectrom 2009; 20:667-73). Separation of extracted proteins is performed by 2D cation exchange LCMS with high-throughput electron capture dissociation (ECD) analysis to identify post translational modification of IR induced protein biomarkers; modifying sublimation matrix application by varying time, temperature and distance between the matrix powder platform and the mounted tissue, FIG. 16; thinner tissue sections with a high performance cryostat; and complete removal of tissue embedding media polymer residue by varying solvent baths.
The application of matrix assisted laser distortion electro-spray ionization Fourier transform ion cyclotron mass spectrometry, FTICR, for both liquid samples and imaging, MALDESI imaging, will improve the isotopic resolution by 2 orders of magnitude. The MALDI system full width half mass, FWHM, isotopic resolution is 2500 Da. The MALDESI system full width half mass, FWHM, isotopic resolution is 250,000 Da. The spatial resolution of MALDI images will improve with laser M2 beam quality closer to 1, increased repetition rate, Hz, fluence, mj/s (Watts), Energy, E, mj/pulse, pulse width, increased work function, smaller spot size, and, optics (beam focus); and decremental sample stage movement from microns to nanometers.
The following Table 6 provides examples of other potential biomarkers of ionizing radiation isolated from buccal tissue in subsequent examples obtained according to the procedures set forth in Example 3.







TABLE 6







K1C10_MOUSE


Keratin, type I cytoskeletal 10 OS = Mus musculus GN = Krt10 PE = 1 SV = 3


ALBU_MOUSE


Serum albumin OS = Mus musculus GN = Alb PE = 1 SV = 3


K2C75_MOUSE


Keratin, type II cytoskeletal 75 OS = Mus musculus GN = Krt75 PE = 1 SV = 1


K2C1_MOUSE


Keratin, type II cytoskeletal 1 OS = Mus musculus GN = Krt1 PE = 1 SV = 4


K1C14_MOUSE


Keratin, type I cytoskeletal 14 OS = Mus musculus GN = Krt14 PE = 1 SV = 2


K2C5_MOUSE


Keratin, type II cytoskeletal 5 OS = Mus musculus GN = Krt5 PE = 2 SV = 1


K1C15_MOUSE


Keratin, type I cytoskeletal 15 OS = Mus musculus GN = Krt15 PE = 1 SV = 2


K1C13_MOUSE


Keratin, type I cytoskeletal 13 OS = Mus musculus GN = Krt13 PE = 2 SV = 2


K2C6A_MOUSE


Keratin, type II cytoskeletal 6A OS = Mus musculus GN = Krt6a PE = 2 SV = 3


K2C4_MOUSE


Keratin, type II cytoskeletal 4 OS = Mus musculus GN = Krt4 PE = 2 SV = 2


K2C73_MOUSE


Keratin, type II cytoskeletal 73 OS = Mus musculus GN = Krt73 PE = 2 SV = 1


K1C17_MOUSE


Keratin, type I cytoskeletal 17 OS = Mus musculus GN = Krt17 PE = 1 SV = 3


K22O_MOUSE


Keratin, type II cytoskeletal 2 oral OS = Mus musculus GN = Krt76 PE = 2 SV = 1


K2C79_MOUSE


Keratin, type II cytoskeletal 79 OS = Mus musculus GN = Krt79 PE = 2 SV = 2


HBA_MOUSE


Hemoglobin subunit alpha OS = Mus musculus GN = Hba PE = 1 SV = 2


AATC_MOUSE


Aspartate aminotransferase, cytoplasmic OS = Mus musculus GN = Got1 PE = 1 SV = 2


K1C19_MOUSE


Keratin, type I cytoskeletal 19 OS = Mus musculus GN = Krt19 PE = 2 SV = 1


K1C42_MOUSE


Keratin, type I cytoskeletal 42 OS = Mus musculus GN = Krt42 PE = 1 SV = 1


K2C1B_MOUSE


Keratin, type II cytoskeletal 1b OS = Mus musculus GN = Krt77 PE = 1 SV = 1


K2C71_MOUSE


Keratin, type II cytoskeletal 71 OS = Mus musculus GN = Krt71 PE = 1 SV = 1


K2C74_MOUSE


Keratin, type II cytoskeletal 74 OS = Mus musculus GN = Krt74 PE = 2 SV = 1


K2C8_MOUSE


Keratin, type II cytoskeletal 8 OS = Mus musculus GN = Krt8 PE = 1 SV = 4


K1C27_MOUSE


Keratin, type I cytoskeletal 27 OS = Mus musculus GN = Krt27 PE = 1 SV = 1


K2C72_MOUSE


Keratin, type II cytoskeletal 72 OS = Mus musculus GN = Krt72 PE = 2 SV = 1


FABP4_MOUSE


Fatty acid-binding protein, adipocyte OS = Mus musculus GN = Fabp4 PE = 1 SV = 3


PTMS_MOUSE


Parathymosin OS = Mus musculus GN = Ptms PE = 2 SV = 3 


K222P_MOUSE


Keratin-like protein KRT222 OS = Mus musculus GN = Krt222 PE = 2 SV = 1


K22E_MOUSE


Keratin, type II cytoskeletal 2 epidermal OS = Mus musculus GN = Krt2 PE = 1 SV = 1


HHB2_MOUSE


Hemoglobin subunit beta-2 OS = Mus musculus GN = Hbb-b2 PE = 1 SV = 2


TPIS_MOUSE


Triosephosphate isomerase OS = Mus musculus GN = Tpi1 PE = 1 SV = 3


FABPH_MOUSE


Fatty acid-binding protein, heart OS = Mus musculus GN = Fabp3 PE = 1 SV = 5


BICD2_MOUSE


Protein bicaudal D homolog 2 OS = Mus musculus GN = Bicd2 PE = 1 SV = 1


K2C1_PANTR


Keratin, type II cytoskeletal 1 OS = Pan troglodytes GN = KRT1 PE = 2 SV = 1


K2C1_HUMAN


Keratin, type II cytoskeletal 1 OS = Homo sapiens GN = KRT1 PE = 1 SV = 5


K1C9_HUMAN


Keratin, type I cytoskeletal 9 OS = Homo sapiens GN = KRT9 PE = 1 SV = 2


K22E_HUMAN


Keratin, type II cytoskeletal 2 epidermal OS = Homo sapiens GN = KRT2 PE = 1 SV = 1


K1C10_HUMAN


Keratin, type I cytoskeletal 10 OS = Homo sapiens GN = KRT10 PE = 1 SV = 4


K1C10_CANFA


Keratin, type I cytoskeletal 10 OS = Canis familiaris GN = KRT10 PE = 2 SV = 1


K2C1_RAT


Keratin, type II cytoskeletal 1 OS = Rattus norvegicus GN = Krt1 PE = 2 SV = 1


K22E_MOUSE


Keratin, type II cytoskeletal 2 epidermal OS = Mus musculus GN = Krt2 PE = 1 SV = 1


K1C14_HUMAN


Keratin, type I cytoskeletal 14 OS = Homo sapiens GN = KRT14 PE = 1 SV = 3


K1C10_BOVIN


Keratin, type I cytoskeletal 10 OS = Bos taurus GN = KRT10 PE = 3 SV = 1


K2C75_BOVIN


Keratin, type II cytoskeletal 75 OS = Bos taurus GN = KRT75 PE = 2 SV = 1


TRYP_PIG


Trypsin OS = Sus scrofa PE = 1 SV = 1


K2C75_RAT


Keratin, type II cytoskeletal 75 OS = Rattus norvegicus GN = Krt75 PE = 2 SV = 2


K2C6A_RAT


Keratin, type II cytoskeletal 6A OS = Rattus norvegicus GN = Krt6a PE = 1 SV = 1


ALBU_MOUSE


Serum albumin OS = Mus musculus GN = Alb PE = 1 SV = 3


K2C4_RAT


Keratin, type II cytoskeletal 4 OS = Rattus norvegicus GN = Krt4 PE = 2 SV = 1


K2C73_HUMAN


Keratin, type II cytoskeletal 73 OS = Homo sapiens GN = KRT73 PE = 1 SV = 1


K2C6A_HUMAN


Keratin, type II cytoskeletal 6A OS = Homo sapiens GN = KRT6A PE = 1 SV = 3


K2C73_MOUSE


Keratin, type II cytoskeletal 73 OS = Mus musculus GN = Krt73 PE = 2 SV = 1


K2C1B_MOUSE


Keratin, type II cytoskeletal 1b OS = Mus musculus GN = Krt77 PE = 1 SV = 1


K2C5_RAT


Keratin, type II cytoskeletal 5 OS = Rattus norvegicus GN = Krt5 PE = 1 SV = 1


K2C6A_MOUSE 


Keratin, type II cytoskeletal 6A OS = Mus musculus GN = Krt6a PE = 2 SV = 3


K2C4_MOUSE


Keratin, type II cytoskeletal 4 OS = Mus musculus GN = Krt4 PE = 2 SV = 2


K22O_MOUSE


Keratin, type II cytoskeletal 2 oral OS = Mus musculus GN = Krt76 PE = 2 SV = 1


K2C3_HUMAN


Keratin, type II cytoskeletal 3 OS = Homo sapiens GN = KRT3 PE = 1 SV = 2


K2C72_HUMAN


Keratin, type II cytoskeletal 72 OS = Homo sapiens GN = KRT72 PE = 1 SV = 2


HBD_TARSY


Hemoglobin subunit delta OS = Tarsius syrichta GN = HBD PE = 2 SV = 2 


K1C13_MOUSE


Keratin, type I cytoskeletal 13 OS = Mus musculus GN = Krt13 PE = 2 SV = 2


HBA_CRIGA


Hemoglobin subunit alpha OS = Cricetomys gambianus GN = HBA PE = 1 SV = 2


TRY1_RAT


Anionic trypsin-1 OS = Rattus norvegicus GN = Prss1 PE = 1 SV = 1


K2C8_MOUSE


Keratin, type II cytoskeletal 8 OS = Mus musculus GN = Krt8 PE = 1 SV = 4


HBB_CALTO


Hemoglobin subunit beta OS = Callicebus torquatus GN = HBB PE = 2 SV = 3


K1C3_XENLA


Keratin, type I cytoskeletal 47 kDa (Fragment) OS = Xenopus laevis GN = xk81b1


PE = 3 SV = 2


K1C24_HUMAN


Keratin, type I cytoskeletal 24 OS = Homo sapiens GN = KRT24 PE = 1 SV = 1


K2C4_HUMAN


Keratin, type II cytoskeletal 4 OS = Homo sapiens GN = KRT4 PE = 1 SV = 4


AATC_HORSE


Aspartate aminotransferase, cytoplasmic OS = Equus caballus GN = GOT1 PE = 1 SV = 2


K1C16_HUMAN


Keratin, type I cytoskeletal 16 OS = Homo sapiens GN = KRT16 PE = 1 SV = 4


HBA_HUMAN


Hemoglobin subunit alpha OS = Homo sapiens GN = HBA1 PE = 1 SV = 2


HBA1_BUBBU


Hemoglobin subunit alpha-1 OS = Bubalus bubalis PE = 2 SV = 3


K1C42_RAT


Keratin, type I cytoskeletal 42 OS = Rattus norvegicus GN = Krt42 PE = 2 SV = 1


K1C14_CHICK


Keratin, type I cytoskeletal 14 OS = Gallus gallus GN = KRT14 PE = 2 SV = 1


HBA_MESAU


Hemoglobin subunit alpha OS = Mesocricetus auratus GN = HBA PE = 1 SV = 1


UP03_PINHA


Unknown protein 3 (Fragment) OS = Pinus halepensis PE = 1 SV = 1


IGH1M_MOUSE


Ig gamma-1 chain C region, membrane-bound form OS = Mus musculus GN = Ighg1


PE = 1 SV = 2


K2C5_PANTR


Keratin, type II cytoskeletal 5 OS = Pan troglodytes GN = KRT5 PE = 2 SV = 1


HBA_BISBO


Hemoglobin subunit alpha-I/II OS = Bison bonasus PE = 1 SV = 2


HBA_TALEU


Hemoglobin subunit alpha OS = Talpa europaea GN = HBA PE = 1 SV = 1


HIR3_CHAGB


Histone transcription regulator 3 homolog OS = Chaetomium globosum GN = HIR3


PE = 3 SV = 1


K1C10_HUMAN


Keratin, type I cytoskeletal 10 OS = Homo sapiens GN = KRT10 PE = 1 SV = 4


K22E_HUMAN


Keratin, type II cytoskeletal 2 epidermal OS = Homo sapiens GN = KRT2 PE = 1 SV = 1


K1C9_HUMAN


Keratin, type I cytoskeletal 9 OS = Homo sapiens GN = KRT9 PE = 1 SV = 2


K2C1_PANTR


Keratin, type II cytoskeletal 1 OS = Pan troglodytes GN = KRT1 PE = 2 SV = 1


K2C1_HUMAN


Keratin, type II cytoskeletal 1 OS = Homo sapiens GN = KRT1 PE = 1 SV = 5


K1C10_CANFA


Keratin, type I cytoskeletal 10 OS = Canis familiaris GN = KRT10 PE = 2 SV = 1


K1C14_HUMAN


Keratin, type I cytoskeletal 14 OS = Homo sapiens GN = KRT14 PE = 1 SV = 3


K2C5_PANTR


Keratin, type II cytoskeletal 5 OS = Pan troglodytes GN = KRT5 PE = 2 SV = 1


K2C6A_HUMAN


Keratin, type II cytoskeletal 6A OS = Homo sapiens GN = KRT6A PE = 1 SV = 3


K1C16_HUMAN


Keratin, type I cytoskeletal 16 OS = Homo sapiens GN = KRT16 PE = 1 SV = 4


TRYP_PIG


Trypsin OS = Sus scrofa PE = 1 SV = 1


K2C79_HUMAN


Keratin, type II cytoskeletal 79 OS = Homo sapiens GN = KRT79 PE = 1 SV = 1


K1C15_HUMAN


Keratin, type I cytoskeletal 15 OS = Homo sapiens GN = KRT15 PE = 1 SV = 2


K1C15_SHEEP


Keratin, type I cytoskeletal 15 OS = Ovis aries GN = KRT15 PE = 2 SV = 1


K1C10_BOVIN


Keratin, type I cytoskeletal 10 OS = Bos taurus GN = KRT10 PE = 3 SV = 1


K1C13_HUMAN


Keratin, type I cytoskeletal 13 OS = Homo sapiens GN = KRT13 PE = 1 SV = 3


K2CO_CHICK


Keratin, type II cytoskeletal cochleal OS = Gallus gallus PE = 2 SV = 1


K2C1_RAT


Keratin, type II cytoskeletal 1 OS = Rattus norvegicus GN = Krt1 PE = 2 SV = 1


K2C5_RAT


Keratin, type II cytoskeletal 5 OS = Rattus norvegicus GN = Krt5 PE = 1 SV = 1


K2C75_BOVIN


Keratin, type II cytoskeletal 75 OS = Bos taurus GN = KRT75 PE = 2 SV = 1


K2C7_BOVIN


Keratin, type II cytoskeletal 7 OS = Bos taurus GN = KRT7 PE = 2 SV = 1


K2C5_BOVIN


Keratin, type II cytoskeletal 5 OS = Bos taurus GN = KRT5 PE = 1 SV = 1


K2C6A_RAT


Keratin, type II cytoskeletal 6A OS = Rattus norvegicus GN = Krt6a PE = 1 SV = 1


K2C75_RAT


Keratin, type II cytoskeletal 75 OS = Rattus norvegicus GN = Krt75 PE = 2 SV = 2


K2C73_MOUSE


Keratin, type II cytoskeletal 73 OS = Mus musculus GN = Krt73 PE = 2 SV = 1


K22E_CANFA


Keratin, type II cytoskeletal 2 epidermal OS = Canis familiaris GN = KRT2 PE = 2


SV = 1


K2C4_RAT


Keratin, type II cytoskeletal 4 OS = Rattus norvegicus GN = Krt4 PE = 2 SV = 1 


K1C3_XENLA


Keratin, type I cytoskeletal 47 kDa (Fragment) OS = Xenopus laevis GN = xk81b1


PE = 3 SV = 2


K22O_HUMAN


Keratin, type II cytoskeletal 2 oral OS = Homo sapiens GN = KRT76 PE = 1 SV = 1


K2C3_RABIT


Keratin, type II cytoskeletal 3 OS = Oryctolagus cuniculus GN = KRT3 PE = 2 SV = 1


K1C19_RAT


Keratin, type I cytoskeletal 19 OS = Rattus norvegicus GN = Krt19 PE = 1 SV = 2


ALBU_MOUSE


Serum albumin OS = Mus musculus GN = Alb PE = 1 SV = 3


K2C8_XENLA


Keratin, type II cytoskeletal 8 OS = Xenopus laevis PE = 2 SV = 1


K2C8_RAT


Keratin, type II cytoskeletal 8 OS = Rattus norvegicus GN = Krt8 PE = 1 SV = 3


K22O_MOUSE


Keratin, type II cytoskeletal 2 oral OS = Mus musculus GN = Krt76 PE = 2 SV = 1


K2C4_HUMAN


Keratin, type II cytoskeletal 4 OS = Homo sapiens GN = KRT4 PE = 1 SV = 4


TRY1_RAT


Anionic trypsin-1 OS = Rattus norvegicus GN = Prssl PE = 1 SV = 1


K1C13_MOUSE


Keratin, type I cytoskeletal 13 OS = Mus musculus GN = Krt13 PE = 2 SV = 2


K2C4_MOUSE


Keratin, type II cytoskeletal 4 OS = Mus musculus GN = Krt4 PE = 2 SV = 2


K2C8_DANRE


Keratin, type II cytoskeletal 8 OS = Danio rerio GN = krt8 PE = 1 SV = 1


KRT85_MOUSE


Keratin, type II cuticular Hb5 OS = Mus musculus GN = Krt85 PE = 2 SV = 2


K2C80_SOVIN


Keratin, type II cytoskeletal 80 OS = Bos taurus GN = KRT80 PE = 2 SV = 1


K1C12_RABIT


Keratin, type I cytoskeletal 12 (Fragment) OS = Oryctolagus cuniculus


GN = KRT12 PE = 2 SV = 1


K1C12_RAT


Keratin, type I cytoskeletal 12 OS = Rattus norvegicus GN = Krt12 PE = 2 SV = 1


K2C1B_RAT


Keratin, type II cytoskeletal 1b OS = Rattus norvegicus GN = Krt77 PE = 2 SV = 1


K1C1_XENLA


Keratin, type I cytoskeletal 47 kDa OS = Xenopus laevis GN = xk81a1 PE = 2 SV = 1


K2C73_HUMAN


Keratin, type II cytoskeletal 73 OS = Homo sapiens GN = KRT73 PE = 1 SV = 1


K1C24_HUMAN


Keratin, type I cytoskeletal 24 OS = Homo sapiens GN = KRT24 PE = 1 SV = 1


K2C8_MOUSE


Keratin, type II cytoskeletal 8 OS = Mus musculus GN = Krt8 PE = 1 SV = 4


K2C78_HUMAN


Keratin, type II cytoskeletal 78 OS = Homo sapiens GN = KRT78 PE = 1 SV = 2


K2C72_HUMAN


Keratin, type II cytoskeletal 72 OS = Homo sapiens GN = KRT72 PE = 1 SV = 2


K2C72_MOUSE


Keratin, type II cytoskeletal 72 OS = Mus musculus GN = Krt72 PE = 2 SV = 1


UP01_PINHA 


Unknown protein 1 (Fragment) OS = Pinus halepensis PE = 1 SV = 1


K22E_MOUSE


Keratin, type II cytoskeletal 2 epidermal OS = Mus musculus GN = Krt2 PE = 1 SV = 1


K1C14_CHICK


Keratin, type I cytoskeletal 14 OS = Gallus gallus GN = KRT14 PE = 2 SV = 1 


K1C27_HUMAN


Keratin, type I cytoskeletal 27 OS = Homo sapiens GN = KRT27 PE = 1 SV = 1


UP03_PINHA


Unknown protein 3 (Fragment) OS = Pinus halepensis PE = 1 SV = 1


FDL30_ARATH


Putative F-box/FBD/LRR-repeat protein At5g22610 OS = Arabidopsis thaliana


GN = At5g22610 PE = 4 SV = 1


NDC80_CANAL


Probable kinetochore protein NDC80 OS = Candida albicans GN = NDC80 PE = 3 SV = 1


K1C10_HUMAN


Keratin, type I cytoskeletal 10 OS = Homo sapiens GN = KRT10 PE = 1 SV = 4


K22E_HUMAN


Keratin, type II cytoskeletal 2 epidermal OS = Homo sapiens GN = KRT2 PE = 1 SV = 1


K2C1_PANTR


Keratin, type II cytoskeletal 1 OS = Pan troglodytes GN = KRT1 PE = 2 SV = 1


K1C10_CANFA


Keratin, type I cytoskeletal 10 OS = Canis familiaris GN = KRT10 PE = 2 SV = 1


K1C9_HUMAN


Keratin, type I cytoskeletal 9 OS = Homo sapiens GN = KRT9 PE = 1 SV = 2


K2C1_HUMAN


Keratin, type II cytoskeletal 1 OS = Homo sapiens GN = KRT1 PE = 1 SV = 5


K2C75_BOVIN


Keratin, type II cytoskeletal 75 OS = Bos taurus GN = KRT75 PE = 2 SV = 1


K2C75_RAT


Keratin, type II cytoskeletal 75 OS = Rattus norvegicus GN = Krt75 PE = 2 SV = 2


K2C1_RAT


Keratin, type II cytoskeletal 1 OS = Rattus norvegicus GN = Krt1 PE = 2 SV = 1


K2C6A_RAT


Keratin, type II cytoskeletal 6A OS = Rattus norvegicus GN = Krt6a PE = 1 SV = 1


K2C6A_HUMAN


Keratin, type II cytoskeletal 6A OS = Homo sapiens GN = KRT6A PE = 1 SV = 3


K2C5_BOVIN


Keratin, type II cytoskeletal 5 OS = Bos taurus GN = KRT5 PE = 1 SV = 1


K2C5_MOUSE


Keratin, type II cytoskeletal 5 OS = Mus musculus GN = KrtS PE = 2 SV = 1


K2C6A_MOUSE


Keratin, type II cytoskeletal 6A OS = Mus musculus GN = Krt6a PE = 2 SV = 3


K2C79_BOVIN


Keratin, type II cytoskeletal 79 OS = Bos taurus GN = KRT79 PE = 2 SV = 1


ALBU_MOUSE


Serum albumin OS = Mus musculus GN = Alb PE = 1 SV = 3


K2C1_MOUSE


Keratin, type II cytoskeletal 1 OS = Mus musculus GN = Krt1 PE = 1 SV = 4


TRYP_PIG


Trypsin OS = Sus scrofa PE = 1 SV = 1


K1C10_BOVIN


Keratin, type I cytoskeletal 10 OS = Bos taurus GN = KRT10 PE = 3 SV = 1


K22E_MOUSE


Keratin, type II cytoskeletal 2 epidermal OS = Mus musculus GN = Krt2 PE = 1 SV = 1


HBA_SPAEH


Hemoglobin subunit alpha OS = Spalax leucodon ehrenbergi GN = HBA PE = 1 SV = 1


HBA_SPECI


Hemoglobin subunit alpha OS = Spermophilus citellus GN = HBA PE = 1 SV = 1


K2C73_MOUSE


Keratin, type II cytoskeletal 73 OS = Mus musculus GN = Krt73 PE = 2 SV = 1


K2C1B_MOUSE


Keratin, type II cytoskeletal 1b OS = Mus musculus GN = Krt77 PE = 1 SV = 1


K2C4_RAT


Keratin, type II cytoskeletal 4 OS = Rattus norvegicus GN = Krt4 PE = 2 SV = 1


K2C73_HUMAN


Keratin, type II cytoskeletal 73 OS = Homo sapiens GN = KRT73 PE = 1 SV = 1


K2C5_PANTR


Keratin, type II cytoskeletal 5 OS = Pan troglodytes GN = KRT5 PE = 2 SV = 1


TRY1_RAT


Anionic trypsin-1 OS = Rattus norvegicus GN = Prss1 PE = 1 SV = 1


K2C4_MOUSE


Keratin, type II cytoskeletal 4 OS = Mus musculus GN = Krt4 PE = 2 SV = 2


K22O_MOUSE


Keratin, type II cytoskeletal 2 oral OS = Mus musculus GN = Krt76 PE = 2 SV = 1


K2C3_HUMAN


Keratin, type II cytoskeletal 3 OS = Homo sapiens GN = KRT3 PE = 1 SV = 2


K2C72_HUMAN


Keratin, type II cytoskeletal 72 OS = Homo sapiens GN = KRT72 PE = 1 SV = 2


K2C72_MOUSE


Keratin, type II cytoskeletal 72 OS = Mus musculus GN = Krt72 PE = 2 SV = 1


HBA_MOUSE


Hemoglobin subunit alpha OS = Mus musculus GN = Hba PE = 1 SV = 2


K1C14_HUMAN


Keratin, type I cytoskeletal 14 OS = Homo sapiens GN = KRT14 PE = 1 SV = 3


K1C3_XENLA


Keratin, type I cytoskeletal 47 kDa (Fragment) OS = Xenopus laevis GN = xk81b1


PE = 3 SV = 2


K1C1_XENLA


Keratin, type I cytoskeletal 47 kDa OS = Xenopus laevis GN = xk81a1 PE = 2 SV = 1


K1C12_RABIT


Keratin, type I cytoskeletal 12 (Fragment) OS = Oryctolagus cuniculus


GN = KRT12 PE = 2 SV = 1


K1C12_RAT


Keratin, type I cytoskeletal 12 OS = Rattus norvegicus GN = Krt12 PE = 2 SV = 1


K2C8_MOUSE


Keratin, type II cytoskeletal 8 OS = Mus musculus GN = Krt8 PE = 1 SV = 4


ALBU_FELCA


Serum albumin OS = Felis silvestris catus GN = ALB PE = 1 SV = 1


K2C4_HUMAN


Keratin, type II cytoskeletal 4 OS = Homo sapiens GN = KRT4 PE = 1 SV = 4


HBB_CALTO


Hemoglobin subunit beta OS = Callicebus torquatus GN = HBB PE = 2 SV = 3


K1C25_BOVIN 


Keratin, type I cytoskeletal 25 OS = Bos taurus GN = KRT25 PE = 2 SV = 1


HBA_MESAU


Hemoglobin subunit alpha OS = Mesocricetus auratus GN = HBA PE = 1 SV = 1


K2C8_BOVIN


Keratin, type II cytoskeletal 8 OS = Bos taurus GN = KRT8 PE = 2 SV = 3


K2C8_RAT


Keratin, type II cytoskeletal 8 OS = Rattus norvegicus GN = Krt8 PE = 1 SV = 3


MATK_ADELA


Maturase K OS = Adesmia lanata GN = matK PE = 3 SV = 1


HBA_BISBO


Hemoglobin subunit alpha-I/II OS = Bison bonasus PE = 1 SV = 2


HBA_TALEU


Hemoglobin subunit alpha OS = Talpa europaea GN = HBA PE = 1 SV = 1


K1C10_HUMAN


Keratin, type I cytoskeletal 10 OS = Homo sapiens GN = KRT10 PE = 1 SV = 4


K1C9_HUMAN


Keratin, type I cytoskeletal 9 OS = Homo sapiens GN = KRT9 PE = l SV = 2


K1C10_CANFA


Keratin, type I cytoskeletal 10 OS = Canis familiaris GN = KRT10 PE = 2 SV = 1


K22E_HUMAN


Keratin, type II cytoskeletal 2 epidermal OS = Homo sapiens GN = KRT2 PE = 1 SV = 1


K2C1_HUMAN


Keratin, type II cytoskeletal 1 OS = Homo sapiens GN = KRT1 PE = 1 SV = 5


K2C1_PANTR


Keratin, type II cytoskeletal 1 OS = Pan troglodytes GN = KRT1 PE = 2 SV = 1


CASQ1_MOUSE


Calsequestrin-1 OS = Mus musculus GN = Casq1 PE = 2 SV = 2


K2C6A_HUMAN


Keratin, type II cytoskeletal 6A OS = Homo sapiens GN = KRT6A PE = 1 SV = 3


HBB_GORGO


Hemoglobin subunit beta OS = Gorilla gorilla gorilla GN = HBB PE = 1 SV = 2


K2C5_HUMAN


Keratin, type II cytoskeletal 5 OS = Homo sapiens GN = KRT5 PE = 1 SV = 3


K2C1_RAT


Keratin, type II cytoskeletal 1 OS = Rattus norvegicus GN = Krt1 PE = 2 SV = 1


K1C10_BOVIN


Keratin, type I cytoskeletal 10 OS = Bos taurus GN = KRT10 PE = 3 SV = 1


HBB_MELME


Hemoglobin subunit beta OS = Meles meles GN = HBB PE = 1 SV = 1


HBB_CALTO


Hemoglobin subunit beta OS = Callicebus torquatus GN = HBB PE = 2 SV = 3


HBA_HUMAN


Hemoglobin subunit alpha OS = Homo sapiens GN = HBA1 PE = 1 SV = 2


ALBU_RAT 


Serum albumin OS = Rattus norvegicus GN = Alb PE = 1 SV = 2


ALBU_FELCA


Serum albumin OS = Felis silvestris catus GN = ALB PE = 1 SV = 1


K2C6A_RAT


Keratin, type II cytoskeletal 6A OS = Rattus norvegicus GN = Krt6a PE = 1 SV = 1


ALBU_HUMAN


Serum albumin OS = Homo sapiens GN = ALB PE = 1 SV = 2


TRYP_PIG


Trypsin OS = Sus scrofa PE = 1 SV = 1


K2C75_BOVIN


Keratin, type II cytoskeletal 75 OS = Bos taurus GN = KRT75 PE = 2 SV = 1


MLE1_HUMAN


Myosin light chain 1, skeletal muscle isoform OS = Homo sapiens GN = MYL1 PE = 1


SV = 3


ACTS_ATRMM


Actin, alpha skeletal muscle OS = Atractaspis microlepidota microlepidota


GN = ACTA1 PE = 2 SV = 1


ACT2_MOLOC


Actin, muscle-type OS = Molgula oculata PE = 3 SV = 1


HBB_NASNA


Hemoglobin subunit beta OS = Nasua nasua GN = HBB PE = 1 SV = 1


HBB_CANFA


Hemoglobin subunit beta OS = Canis familiaris GN = HBB PE = 1 SV = 1 


HBA_MESAU


Hemoglobin subunit alpha OS = Mesocricetus auratus GN = HBA PE = 1 SV = 1


K2C5_BOVIN


Keratin, type II cytoskeletal 5 OS = Bos taurus GN = KRT5 PE = 1 SV = 1


K2C6A_MOUSE


Keratin, type II cytoskeletal 6A OS = Mus musculus GN = Krt6a PE = 2 SV = 3


HBB_MACMU


Hemoglobin subunit beta OS = Macaca mulatta GN = HBB PE = 1 SV = 1


HBB_SAISC


Hemoglobin subunit beta OS = Saimiri sciureus GN = HBB PE = 1 SV = 2


TRY1_RAT


Anionic trypsin-1 OS = Rattus norvegicus GN = Prss1 PE = 1 SV = 1


K2C8_MOUSE


Keratin, type II cytoskeletal 8 OS = Mus musculus GN = Krt8 PE = 1 SV = 4


K2C1B_RAT


Keratin, type II cytoskeletal 1b OS = Rattus norvegicus GN = Krt77 PE = 2 SV = 1


MDHM_BOVIN


Malate dehydrogenase, mitochondrial OS = Bos taurus GN = MDH2 PE = 2 SV = 1 


HBA_CRIGA


Hemoglobin subunit alpha OS = Cricetomys gambianus GN = HBA PE = 1 SV = 2


HBA_SPECI


Hemoglobin subunit alpha OS = Spermophilus citellus GN = HBA PE = 1 SV = 1


HBB_PAGLA


Hemoglobin subunit beta OS = Paguma larvata GN = HBB PE = 1 SV = 1


HBB_MYOVE


Hemoglobin subunit beta OS = Myotis velifer GN = HBB PE = 1 SV = 1


K1C1_XENLA


Keratin, type I cytoskeletal 47 kDa OS = Xenopus laevis GN = xk81a1 PE = 2 SV = 1


ACT18_DICDI


Actin-18 OS = Dictyostelium discoideum GN = act18 PE = 3 SV = 3


K1C24_HUMAN


Keratin, type I cytoskeletal 24 OS = Homo sapiens GN = KRT24 PE = 1 SV = 1


ACT1_ARTSX


Actin, clone 205 OS = Artemia sp. PE = 2 SV = 1


ACTC_BRAFL


Actin, cytoplasmic OS = Branchiostoma floridae PE = 2 SV = 1


ACTB3_FUGRU


Actin, cytoplasmic 3 OS = Fugu rubripes GN = actbc PE = 2 SV = 1


ACT_THELA


Actin OS = Thermomyces lanuginosus PE = 3 SV = 1


ACT1_SACKO


Actin-1 OS = Saccoglossus kowalevskii PE = 2 SV = 1


ACT1_LYTPI


Actin, cytoskeletal 1 OS = Lytechinus pictus PE = 2 SV = 1


ACTC_BRALA


Actin, cytoplasmic OS = Branchiostoma lanceolatum PE = 2 SV = 1


ACT_CANGA


Actin OS = Candida glabrata GN = ACT1 PE = 3 SV = 1


ACT1_KLULA


Actin OS = Kluyveromyces lactis GN = ACT PE = 3 SV = 2


ACT_PHARH


Actin OS = Phaffia rhodozyma PE = 3 SV = 1


ACT_CANAL


Actin OS = Candida albicans GN = ACT1 PE = 3 SV = 1


ACT_SCHPO


Actin OS = Schizosaccharomyces pombe GN = act1 PE = 1 SV = 1 


ACTBL_HUMAN


Beta-actin-like protein 2 OS = Homo sapiens GN = ACTBL2 PE = 1 SV = 2


A26CA_HUMAN


ANKRD26-like family C member 1A OS = Homo sapiens GN = A26C1A PE = 1 SV = 3


K2C4_RAT


Keratin, type II cytoskeletal 4 OS = Rattus norvegicus GN = Krt4 PE = 2 SV = 1


K2C73_HUMAN


Keratin, type II cytoskeletal 73 OS = Homo sapiens GN = KRT73 PE = 1 SV = 1


HBA_MACFA


Hemoglobin subunit alpha-A/Q/R/T OS = Macaca fascicularis PE = 1 SV = 1


HED_GORGO


Hemoglobin subunit delta OS = Gorilla gorilla gorilla GN = HBD PE = 1 SV = 2


MDHM_YEAST


Malate dehydrogenase, mitochondrial OS = Saccharomyces cerevisiae GN = MDH1


PE = 1 SV = 2


HBA_LAMGL


Hemoglobin subunit alpha OS = Lama glama GN = HBA PE = 1 SV = 1


HBB_RABIT


Hemoglobin subunit beta-1⁄2 OS = Oryctolagus cuniculus GN = HBB1 PE = 1 SV = 2


HED_ELEMA


Hemoglobin subunit delta OS = Elephas maximus GN = HBD PE = 2 SV = 3


FIBB_HUMAN


Fibrinogen beta chain OS = Homo sapiens GN = FGB PE = 1 SV = 2


HED_COLPO


Hemoglobin subunit delta OS = Colobus polykomos GN = HBD PE = 2 SV = 2


K2C4_MOUSE


Keratin, type II cytoskeletal 4 OS = Mus musculus GN = Krt4 PE = 2 SV = 2


K22O_MOUSE


Keratin, type II cytoskeletal 2 oral OS = Mus musculus GN = Krt76 PE = 2 SV = 1


K2C3_RABIT


Keratin, type II cytoskeletal 3 OS = Oryctolagus cuniculus GN = KRT3 PE = 2 SV = 1


TPISA_DANRE


Triosephosphate isomerase A OS = Danio rerio GN = tpi1a PE = 2 SV = 1


TPISA_SPOR


Triosephosphate isomerase OS = Aspergillus oryzae GN = tpiA PE = 2 SV = 1


TPIS_HERAR


Triosephosphate isomerase OS = Herminiimonas arsenicoxydans GN = tpiA PE = 3 SV = 1


VIM4_XENLA


Vimentin-4 OS = Xenopus laevis GN = vim4 PE = 2 SV = 1


VIME_BOVIN


Vimentin OS = Bos taurus GN = VIM PE = 1 SV = 3


MATK_ADELA


Maturase K OS = Adesmia lanata GN = matK PE = 3 SV = 1


CASA1_BOVIN


Alpha-S1-casein OS = Bos taurus GN = CSN1S1 PE = 1 SV = 2


MLE1_CHICK


Myosin light chain 1, skeletal muscle isoform OS = Gallus gallus PE = 1 SV = 3


HBA_ARAAR


Hemoglobin subunit alpha-A OS = Ara ararauna GN = HBAA PE = 1 SV = 2


PYRB_AERHH


Aspartate carbamoyltransferase OS = Aeromonas hydrophila subsp. hydrophila


(strain ATCC 7966 / NCIB 9240) GN = pyrB PE = 3 SV = 2


ATPB_BOVIN


ATP synthase subunit beta, mitochondrial OS = Bos taurus GN = ATP5B PE = 1 SV = 2


Q8BGZ7_MOUSE


10 days neonate skin cDNA, RIKEN full-length enriched library,


clone: 4732475I03 product: CYTOKERATIN homolog (10 days neonate skin cDNA,


RIKEN full-length enriched library, clone: 4732468K03 product: CYTOKERATIN


TYPE II homolog) (6 days neonate skin cD


K2C1_MOUSE


Keratin, type II cytoskeletal 1 (Cytokeratin-1) (CK-1) (Keratin-1) (K1) (67


kDa cytokeratin). - Mus musculus (Mouse).


Q9D2K8_MOUSE


0 day neonate head cDNA, RIKEN full-length enriched library,


clone: 4833436C19 product: keratin complex 2, basic, gene 1, full insert


sequence. - Mus musculus (Mouse).


Q32P04_MOUSE


Krt2-5 protein (Fragment). - Mus musculus (Mouse).


K2C6A_MOUSE


Keratin, type II cytoskeletal 6A (Cytokeratin-6A) (CK 6A) (K6a keratin)


(Keratin-6 alpha) (mK6-alpha). - Mus musculus (Mouse).


Q80VP7_MOUSE


Hypothetical protein MGC54654. - Mus musculus (Mouse).


Q8BIS2_MOUSE


10 days neonate skin cDNA, RIKEN full-length enriched library,


clone: 4732456N10 product: similar to KERATIN, TYPE II CYTOSKELETAL 6D


(CYTOKERATIN 6D) (CK 6D) (K6D KERATIN). - Mus musculus (Mouse).


Q9CXH5_MOUSE


17 days embryo head cDNA, RIKEN full-length enriched library,


clone: 3300001P16 product: hemoglobin, beta adult major chain, full insert


sequence. - Mus musculus (Mouse). 


KRMSE1


keratin, 59K type I cytoskeletal - mouse


Q6NXH9_MOUSE


Type II keratin Kb36. - Mus musculus (Mouse).


K2C1B_MOUSE


Keratin, type II cytoskeletal 1b (Type II keratin Kb39) (Embryonic type II


keratin-1). - Mus musculus (Mouse).


Q6IFZ9_MOUSE


Type II keratin Kb37. - Mus musculus (Mouse).


Q3TTY5_MOUSE


10 days neonate skin cDNA, RIKEN full-length enriched library,


clone: 4732404G19 product: keratin complex 2, basic, gene 17, full insert


sequence (10 days neonate skin cDNA, RIKEN full-length enriched library,


clone: 4732426A12 product: keratin complex


Q6IFT3_MOUSE


Keratin Kb40. - Mus musculus (Mouse).


Q3UV17_MOUSE


Adult female vagina cDNA, RIKEN full-length enriched library,


clone: 9930024P18 product: similar to Keratin 2p. - Mus musculus (Mouse).


I59009


epidermal keratin subunit II - mouse


HAMS


hemoglobin alpha chains - mouse


K2C4_MOUSE


Keratin, type II cytoskeletal 4 (Cytokeratin-4) (CK-4) (Keratin-4) (K4)


(Cytoskeletal 57 kDa keratin). - Mus musculus (Mouse). 


Q6IME9_MOUSE


Type-II keratin Kb35. - Mus musculus (Mouse).


K2C8_MOUSE


Keratin, type II cytoskeletal 8 (Cytokeratin-8) (CK-8) (Keratin-8) (K8)


(Cytokeratin endo A). - Mus musculus (Mouse).


Q3TV03_MOUSE


Adult male stomach cDNA, RIKEN full-length enriched library,


clone: 2210414C06 product: albumin 1, full insert sequence. - Mus musculus


(Mouse).


Q9Z1R9_MOUSE


Trypsinogen 16 (Protease, serine, 1). - Mus musculus (Mouse).


JQ0028


cytokeratin 19 - mouse


Q2M1G8_MOUSE .


2410039E07Rik protein (Fragment). - Mus musculus (Mouse).


AAH11074


BC011074 NID: - Mus musculus


KRMSE1


keratin, 59K type I cytoskeletal - mouse


Q9CY54_MOUSE


13 days embryo liver cDNA, RIKEN full-length enriched library,


clone: 2500004H04 product: hemoglobin, beta adult major chain, full insert


sequence. - Mus musculus (Mouse).


HAMS


hemoglobin alpha chains - mouse


Q9CY06_MOUSE


13 days embryo liver cDNA, RIKEN full-length enriched library,


clone: 2510040P05 product: hemoglobin, beta adult major chain, full insert


sequence. - Mus musculus (Mouse).


AAH11074


BC011074 NID: - Mus musculus


HBMS


hemoglobin beta major chain - mouse


Q3TTY5_MOUSE


10 days neonate skin cDNA, RIKEN full-length enriched library,


clone: 4732404G19 product: keratin complex 2, basic, gene 17, full insert


sequence (10 days neonate skin cDNA, RIKEN full-length enriched library,


clone: 4732426A12 product: keratin complex


Q3UJH8_MOUSE


16 days embryo heart cDNA, RIKEN full-length enriched library,


clone: i920001F12 product: glutamate oxaloacetate transaminase 1, soluble,


full insert sequence. - Mus musculus (Mouse).


Q8BGZ7_MOUSE


10 days neonate skin cDNA, RIKEN full-length enriched library,


clone: 4732475I03 product: CYTOKERATIN homolog (10 days neonate skin cDNA,


RIKEN full-length enriched library, clone: 4732468K03 product: CYTOKERATIN


TYPE II homolog) (6 days neonate skin cD 


K1C17_MOUSE


Keratin, type I cytoskeletal 17 (Cytokeratin-17) (CK-17) (Keratin-17)


(K17). - Mus musculus (Mouse).


Q9R0S6_MOUSE


Beta-1-globin (Fragment). - Mus musculus (Mouse).


K2C1_MOUSE


Keratin, type II cytoskeletal 1 (Cytokeratin-1) (CK-1) (Keratin-1) (K1) (67


kDa cytokeratin). - Mus musculus (Mouse).


Q9D2K8_MOUSE


0 day neonate head cDNA, RIKEN full-length enriched library,


clone: 4833436C19 product: keratin complex 2, basic, gene 1, full insert


sequence. - Mus musculus (Mouse).


A55682


keratin 13, type I cytoskeletal - mouse


Q32P04_MOUSE


Krt2-5 protein (Fragment). - Mus musculus (Mouse).


CAA33084


MMHFABP NID: - Mus musculus


Q80VP7_MOUSE


Hypothetical protein MGC54654. - Mus musculus (Mouse).


Q8BIS2_MOUSE


10 days neonate skin cDNA, RIKEN full-length enriched library,


clone: 4732456N10 product: similar to KERATIN, TYPE II CYTOSKELETAL 6D


(CYTOKERATIN 6D) (CK 6D) (K6D KERATIN). - Mus musculus (Mouse).


Q6NXH9_M


Type II keratin Kb36. - Mus musculus (Mouse). 


OUSE


K1C15_MOUSE


Keratin, type I cytoskeletal 15 (Cytokeratin-15) (CK-15) (Keratin-15)


(K15). - Mus musculus (Mouse).


FABPH_MOUSE


Fatty acid-binding protein, heart (H-FABP) (Heart-type fatty acid- binding


protein) (Mammary-derived growth inhibitor) (MDGI). - Mus musculus (Mouse).


Q3UV17_MOUSE


Adult female vagina cDNA, RIKEN full-length enriched library,


clone: 9930024P18 product: similar to Keratin 2p. - Mus musculus (Mouse).


K2C4_MOUSE


Keratin, type II cytoskeletal 4 (Cytokeratin-4) (CK-4) (Keratin-4) (K4)


(Cytoskeletal 57 kDa keratin). - Mus musculus (Mouse).


Q6IME9_MOUSE


Type-II keratin Kb35. - Mus musculus (Mouse).


Q6IFT3_MOUSE


Keratin Kb40. - Mus musculus (Mouse).


Q8VCW2_MOUSE


RIKEN cDNA 4631426H08. - Mus musculus (Mouse).


Q2M1G8_MOUSE


2410039E07Rik protein (Fragment). - Mus musculus (Mouse).


FABPA_MO


Fatty acid-binding protein, adipocyte (AFABP) (Adipocyte lipid-binding


protein) (ALBP) (A-FABP) (P2 adipocyte protein) (Myelin P2 protein homolog)


(3T3-L1 lipid-binding protein) (422 protein) (P15).-


USE


Mus musculus (Mouse).


I59009


epidermal keratin subunit II - mouse


Q3TV03_MOUSE


Adult male stomach cDNA, RIKEN full-length enriched library,


clone: 2210414C06 product: albumin 1, full insert sequence. - Mus musculus


(Mouse).


DEMSMC


malate dehydrogenase (EC 1.1.1.37), cytosolic - mouse


JQ0028


cytokeratin 19 - mouse


Q3UDM1_MOUSE


Bone marrow macrophage cDNA, RIKEN full-length enriched library,


clone: G530119K21 product: ATP-binding cassette, sub-family C (CFTR/MRP),


member 1, full insert sequence. (Fragment). - Mus musculus (Mouse).


Q3UBW7_MOUSE


Bone marrow macrophage cDNA, RIKEN full-length enriched library,


clone: I830015F18 product: transferrin, full insert sequence. - Mus musculus


(Mouse).


K1C10_MOUSE


Keratin, type I cytoskeletal 10 OS = Mus musculus GN = Krt10 PE = 1 SV = 3


K2C75_MOUSE


Keratin, type II cytoskeletal 75 OS = Mus musculus GN = Krt75 PE = 1 SV = 1


ALBU_MOUSE


Serum albumin OS = Mus musculus GN-Alb PE = 1 SV = 3


K2C5_MOUSE


Keratin, type II cytoskeletal 5 OS = Mus musculus GN = Krt5 PE = 2 SV = 1


K2C1_MOUSE


Keratin, type II cytoskeletal 1 OS = Mus musculus GN = Krt1 PE = 1 SV = 4


K2C6A_MOUSE


Keratin, type II cytoskeletal 6A OS = Mus musculus GN = Krt6a PE = 2 SV = 3


K2C6B_MOUSE


Keratin, type II cytoskeletal 6B OS = Mus musculus GN-Krt6b PE = 2 SV = 3


K2C79_MOUSE


Keratin, type II cytoskeletal 79 OS = Mus musculus GN = Krt79 PE = 2 SV = 2


K1C13_MOUSE


Keratin, type I cytoskeletal 13 OS = Mus musculus GN = Krt13 PE = 2 SV = 2


AATC_MOUSE


Aspartate aminotransferase, cytoplasmic OS = Mus musculus GN = Got1 PE = 1 SV = 2


K1C14_MOUSE


Keratin, type I cytoskeletal 14 OS = Mus musculus GN-Krt14 PE = 1 SV = 2


K1C17_MOUSE


Keratin, type I cytoskeletal 17 OS = Mus musculus GN = Krt17 PE = 1 SV = 3


K2C73_MOUSE


Keratin, type II cytoskeletal 73 OS = Mus musculus GN = Krt73 PE = 2 SV = 1


HBB1_MOUSE


Hemoglobin subunit beta-1 OS = Mus musculus GN = Hbb-b1 PE = 1 SV = 2


K2C71_MOUSE


Keratin; type II cytoskeletal 71 OS = Mus musculus GN = Krt71 PE = 1 SV = 1


K2C4_MOUSE


Keratin, type II cytoskeletal 4 OS = Mus musculus GN = Krt4 PE = 2 SV = 2


K22O_MOUSE


Keratin, type II cytoskeletal 2 oral OS = Mus musculus GN = Krt76 PE = 2 SV = 1


K2C72_MOUSE


Keratin, type II cytoskeletal 72 OS = Mus musculus GN = Krt72 PE = 2 SV = 1


K2C8_MOUSE


Keratin, type II cytoskeletal 8 OS = Mus musculus GN = Krt8 PE = 1 SV = 4


FABPH_MOUSE


Fatty acid-binding protein, heart OS = Mus musculus GN = Fabp3 PE = 1 SV = 5


SOX1_MOUSE


SOX-1 protein OS = Mus musculus GN-Sox1 PE = 2 SV = 1


SPA3M_MOUSE


Serine protease inhibitor A3M OS = Mus musculus GN = Serpina3m PE = 1 SV = 1


TPIS_MOUSE


Triosephosphate isomerase OS = Mus musculus GN = Tpi1 PE = 1 SV = 3


K1C15_MOUSE


Keratin, type I cytoskeletal 15 OS = Mus musculus GN = Krt15 PE = 1 SV = 2


K1C16_MOUSE


Keratin, type I cytoskeletal 16 OS = Mus musculus GN = Krt16 PE = 1 SV = 3


K22E_MOUSE


Keratin, type II cytoskeletal 2 epidermal OS = Mus musculus GN = Krt2 PE = 1 SV = 1


DNJC1_MOUSE


DnaJ homolog subfamily C member 1 OS = Mus musculus GN = Dnajc1 PE = 1 SV = 1


HBA_MOUSE


Hemoglobin subunit alpha OS = Mus musculus GN = Hba PE = 1 SV = 2


ARVC_MOUSE


Armadillo repeat protein deleted in velo-cardio-facial syndrome homolog


OS = Mus musculus GN = Arvcf PE = 1 SV = 2


VIME_MOUSE


Vimentin OS = Mus musculus GN = Vim PE = 1 SV = 3


KRT83_MOUSE


Keratin, type II cuticular Hb3 OS = Mus musculus GN = Krt83 PE = 2 SV = 2


KRT82_MOUSE


Keratin, type II cuticular Hb2 OS = Mus musculus GN = Krt82 PE = 2 SV = 1


A1AT1_MOUSE


Alpha-l-antitrypsin 1-1 OS = Mus musculus GN = Serpinala PE = 1 SV = 4


SODC_MOUSE


Superoxide dismutase [Cu-Zn] OS = Mus musculus GN = Sod1 PE = 1 SV = 2


AS250_MOUSE


250 kDa substrate of Akt OS = Mus musculus GN = Kiaa1272 PE = 1 SV = 2


K1C25_MOUSE


Keratin, type I cytoskeletal 25 OS = Mus musculus GN = Krt25 PE = 1 SV = 1


HBB1_MOUSE


Hemoglobin subunit beta-1 OS = Mus musculus GN = Hbb-bl PE = 1 SV = 2


K1C10_MOUSE


Keratin, type I cytoskeletal 10 OS = Mus musculus GN = Krt10 PE = 1 SV = 3


K2C75_MOUSE


Keratin, type II cytoskeletal 75 OS = Mus musculus GN = Krt75 PE = 1 SV = 1


K2C1_MOUSE


Keratin, type II cytoskeletal 1 OS = Mus musculus GN = Krt1 PE = 1 SV = 4


K2C5_MOUSE


Keratin, type II cytoskeletal 5 OS = Mus musculus GN = Krt5 PE = 2 SV = 1


ALBU_MOUSE


Serum albumin OS = Mus musculus GN-Alb PE = 1 SV = 3


K2C79_MOUSE


Keratin, type II cytoskeletal 79 OS = Mus musculus GN = Krt79 PE = 2 SV = 2


K1C42_MOUSE


Keratin, type I cytoskeletal 42 OS = Mus musculus GN = Krt42 PE = 1 SV = 1


HBA_MOUSE


Hemoglobin subunit alpha OS = Mus musculus GN = Hba PE = 1 SV = 2


K2C73_MOUSE


Keratin, type II cytoskeletal 73 OS = Mus musculus GN = Krt73 PE = 2 SV = 1


K1C15_MOUSE


Keratin, type I cytoskeletal 15 OS = Mus musculus GN = Krt15 PE = 1 SV = 2


K2C1B_MOUSE


Keratin, type II cytoskeletal 1b OS = Mus musculus GN = Krt77 PE = 1 SV = 1


K1C27_MOUSE


Keratin, type I cytoskeletal 27 OS = Mus musculus GN = Krt27 PE = 1 SV = 1


K2C4_MOUSE


Keratin; type II cytoskeletal 4 OS = Mus musculus GN = Krt4 PE = 2 SV = 2


K22O_MOUSE


Keratin, type II cytoskeletal 2 oral OS = Mus musculus GN = Krt76 PE = 2 SV = 1


K2C72_MOUSE


Keratin, type II cytoskeletal 72 OS = Mus musculus GN = Krt72 PE = 2 SV = 1


K1C19_MOUSE


Keratin, type I cytoskeletal 19 OS = Mus musculus GN = Krt19 PE = 2 SV = 1


K1C17_MOUSE


Keratin, type I cytoskeletal 17 OS = Mus musculus GN = Krt17 PE = 1 SV = 3


K22E_MOUSE


Keratin, type II cytoskeletal 2 epidermal OS = Mus musculus GN = Krt2 PE = 1 SV = 1


SYTC_MOUSE


Threonyl-tRNA synthetase, cytoplasmic OS = Mus musculus GN = Tars PE = 1 SV = 2


HBE_MOUSE


Hemoglobin subunit epsilon-Y2 OS = Mus musculus GN = Hbb-y PE = 1 SV = 2


FTSJ2_MOUSE


FtsJ methyltransferase domain-containing protein 2 OS = Mus musculus


GN = Ftsjd2 PE = 1 SV = 1


DPOLA_MOUSE


DNA polymerase alpha catalytic subunit OS = Mus musculus GN = Polal PE = 1 SV = 2


HBB1_MOUSE Mass: 15830 Score: 216 Queries matched: 10 emPAI: 2.


Hemoglobin subunit beta-1 OS = Mus musculus GN = Hbb-b1 PE = 1 SV = 2


Check to include this hit in error tolerant search or archive report


HAMS


hemoglobin alpha chains - mouse


Q9CY12_MOUSE


13 days-embryo liver cDNA, RIKEN full-length enriched library,


clone: 2510039D09 product: hemoglobin, beta adult major chain, full insert


sequence. - Mus musculus (Mouse).


Q8BGZ7_MOUSE


10 days neonate skin cDNA, RIKEN full-length enriched library,


clone: 4732475I03 product: CYTOKERATIN homolog (10 days neonate skin cDNA,


RIKEN full-length enriched library, clone: 4732468K03 product: CYTOKERATIN


TYPE II homolog) (6 days neonate skin cD


Q32P04_MOUSE


Krt2-5 protein (Fragment). - Mus musculus (Mouse).


Q3TV03_MOUSE


Adult male stomach cDNA, RIKEN full-length enriched library,


clone: 2210414C06 product: albumin 1, full insert sequence. - Mus musculus


(Mouse).


KRMSE1


keratin, 59K type I cytoskeletal - mouse


K1C15_MOUSE


Keratin, type I cytoskeletal 15 (Cytokeratin-15) (CK-15) (Keratin-15)


(K15). - Mus musculus (Mouse).


Q8BIS2_MOUSE


10 days neonate skin cDNA, RIKEN full-length enriched library,


clone: 4732456N10 product: similar to KERATIN, TYPE II CYTOSKELETAL 6D


(CYTOKERATIN 6D) (CK 6D) (K6D KERATIN). - Mus musculus (Mouse).


Q8BGZ7_MOUSE


10 days neonate skin cDNA, RIKEN full-length enriched library,


clone: 4732475I03 product: CYTOKERATIN homolog (10 days neonate skin cDNA,


RIKEN full-length enriched library, clone: 4732468K03 product: CYTOKERATIN


TYPE II homolog) (6 days neonate skin cD


K2C6A_MOUSE


Keratin, type II cytoskeletal 6A (Cytokeratin-6A). (CK 6A) (K6a keratin)


(Keratin-6 alpha) (mK6-alpha). - Mus musculus (Mouse).


Q32P04_MOUSE


Krt2-5 protein (Fragment). - Mus musculus (Mouse).


Q8K2E4_MOUSE


CDNA sequence BC031593. - Mus musculus (Mouse).


K2C1_MOUSE


Keratin, type II cytoskeletal 1 (Cytokeratin-1) (CK-1) (Keratin-1) (K1) (67


kDa cytokeratin). - Mus musculus (Mouse).


Q9D2K8_MOUSE


0 day neonate head cDNA, RIKEN full-length enriched library,


clone: 4833436C19 product: keratin complex 2, basic, gene 1, full insert


sequence. - Mus musculus (Mouse).


Q8BIS2_MOUSE


10 days neonate skin cDNA, RIKEN full-length enriched library,


clone: 4732456N10 product: similar to KERATIN, TYPE II CYTOSKELETAL 6D


(CYTOKERATIN 6D) (CK 6D) (K6D KERATIN). - Mus musculus (Mouse).


Q80VP7_MOUSE


Hypothetical protein MGC54654. - Mus musculus (Mouse).


Q6NXH9_MOUSE


Type II keratin Kb36. - Mus musculus (Mouse).


Q6IFT3_MO


Keratin Kb40. - Mus musculus (Mouse). 


USE


K2C1B_MOUSE


Keratin, type II cytoskeletal 1b (Type II keratin Kb39) (Embryonic type II


keratin-1). - Mus musculus (Mouse).


Q3UV17_MOUSE


Adult female vagina cDNA, RIKEN full-length enriched library,


clone: 9930024P18 product: similar to Keratin 2p. - Mus musculus (Mouse).


K2C4_MOUSE


Keratin, type II cytoskeletal 4 (Cytokeratin-4) (CK-4) (Keratin-4) (K4)


(Cytoskeletal 57 kDa keratin). - Mus musculus (Mouse).


AAH11074


BC011074 NID: - Mus musculus


BAA85657


AB033744 NID: - Mus musculus


Q6IFZ9_MOUSE


Type II keratin Kb37. - Mus musculus (Mouse).


159009


epidermal keratin subunit II - mouse


K1C17_MOUSE


Keratin, type I cytoskeletal 17 (Cytokeratin-17) (CK-17) (Keratin-17)


(K17). - Mus musculus (Mouse).


Q6IFX2_MOUSE


Type I keratin KA22. - Mus musculus (Mouse).


Q6IME9_MOUSE


Type-II keratin Kb35. - Mus musculus (Mouse).


BAE40567


AK168726 NID: - Mus musculus


AAD01692


AF021836 NID: - Mus musculus


Q3USS4


Adult male corpora quadrigemina cDNA, RIKEN full-length enriched library,


clone: B230315D14 product: glial fibrillary acidic protein, full insert


sequence. - Mus musculus (Mouse). 


_MOUSE


Q3TTY5_MOUSE


10 days neonate skin cDNA, RIKEN full-length enriched library,


clone: 4732404G19 product: keratin complex 2, basic, gene 17, full insert


sequence (10 days neonate skin cDNA, RIKEN full-length enriched library,


clone: 4732426A12 product: keratin complex


K1C15_MOUSE


Keratin, type I cytoskeletal 15 (Cytokeratin-15) (CK-15) (Keratin-15)


(K15). - Mus musculus (Mouse).


Q3TWV0_MOUSE


Osteoclast-like cell cDNA, RIKEN full-length enriched library,


clone: 1420023H06 product: vimentin, full insert sequence. - Mus musculus


(Mouse).


Q9JKB4_MOUSE


Epidermal keratin 10 (Fragment). - Mus musculus (Mouse).


Q8VCW2_MOUSE


RIKEN cDNA 4631426H08. - Mus musculus (Mouse).


JQ0028 


cytokeratin 19 - mouse


KRMSE1


keratin, 59K type I cytoskeletal - mouse


JC4030


DnaJ-like protein MTJ1 - mouse


E2AK4_MOUSE


Eukaryotic translation initiation factor 2-alpha kinase 4 (EC 2.7.11.1)


(GCN2-like protein) (mGCN2). - Mus musculus (Mouse).


BAB27580 


AK011380 NID: - Mus musculus

## Claims

1. A method of quantifying the amount of ionizing radiation to which an individual has been exposed comprising determining the presence of one or more radiation associated markers.

2. A method according to claim 1 wherein the sample is selected from the group consisting of a buccal swab, saliva, plasma, serum, urine and blood.

3. A method according to claim 1 wherein determining the presence of one or more radiation associated markers in a sample is performed by Matrix assisted laser desorption ionization (MALDI) mass spectrometry.

4. A method according to claim 1 wherein determining the presence of one or more radiation associated markers in a sample is performed by (a) contacting the sample with an antibody which specifically binds to a radiation associated marker permitting formation of a complex between the antibody and the radiation associated marker; and (b) measuring the amount of complexes formed, thereby determining the amount of the radiation associated marker in the sample.

5. A method according to claim 4 further comprising (c) comparing the amount of radiation associated marker in the sample determined in step (b) with either (i) the amount determined for temporally matched, normal samples or (ii) the amount determined for samples obtained from individuals who have not been exposed to elevated levels of ionizing radiation.