Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
  • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
  • G01N33/6848—Methods of protein analysis involving mass spectrometry
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
  • C12M1/00—Apparatus for enzymology or microbiology
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
  • G01N33/57407—Specifically defined cancers
  • G01N33/57423—Specifically defined cancers of lung
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
  • G01N33/57407—Specifically defined cancers
  • G01N33/57438—Specifically defined cancers of liver, pancreas or kidney
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2800/00—Detection or diagnosis of diseases
  • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Definitions

• the present invention relates to methods of enhancing the identification of peaks in mass spectra data for use in the early prediction, detection, and response to treatment of diseases in a human.
• the health of a cell and of an organism is reflected by the proteins and other molecules that it contains.
• the detection, identification, and quantification of proteins and other molecules, such as lipids and carbohydrates, may facilitate disease mechanism elucidation, early detection of disease, prediction of disease, and evaluation of treatments.
• a "marker” typically refers to a polypeptide or some other molecule that differentiates one biological status from another.
• Recently developed methods for molecule detection have made it possible to measure a large fraction of these molecules, opening up a range of new, targeted methods for disease detection, prevention, and treatment. To effectively practice such methods requires the ability to identify individual molecules or markers, often at low concentrations, from mixtures of hundreds or thousands of different compounds.
• mass spectrometric formats include matrix assisted laser desorption/ionization mass spectrometry (MALDI), see, e.g., U.S. Pat. No. 5,118,937 and U.S. Pat. No. 5,045,694, and surface enhanced laser desorption/ionization mass spectrometry (SELDI) 5 see, e.g., U.S. Pat. No. 5,719,060.
• MALDI matrix assisted laser desorption/ionization mass spectrometry
• SELDI surface enhanced laser desorption/ionization mass spectrometry
• the great advantage of mass spectrometry over other technologies for global detection and monitoring of subtle changes in cell function is the ability to measure rapidly and inexpensively thousands of elements in a few microliters of biological fluid. For example, disease processes that result from altered genes, such as cancer, produce altered protein products that circulate in the blood as polypeptides and other molecules of varying size. Mass spectrometry allows for the detection of such products and the subsequent diagnosis and analysis of the disease.
• the present invention relates to methods of enhancing the identification of peaks in mass spectra data for use in the early prediction, detection, and response to treatment of diseases in a human.
• One embodiment of the present invention includes a method for determining the probability of disease.
• the method may comprise the steps of filtering a biological fluid through a hole array filter, generating mass spectra data from the filtered biological fluid, and comparing the mass spectra data with a database.
• Yet another embodiment of the present invention includes a method of predicting response to disease treatment.
• the method may comprise the steps of generating a first set of mass spectra data from a first set of samples from a population that responds to a treatment of a disease A after filtration of the first set of samples through a hole array filter and generating a second set of mass spectra data from a second set of samples from a population that does not respond to the same treatment of disease A after filtration ofthe second set of samples through a hole array filter.
• the method may also include the step of comparing corresponding peaks in first and second sets of mass spectra data, wherein a difference in corresponding peaks indicates that the peaks represent at least one marker indicating the likelihood of response to the treatment of disease A.
• the at least one marker may be used to predict the likelihood of response to the treatment of disease.
• the structure ofthe hole array filter through which the first set of samples are filtered and the structure ofthe hole array filter through which the second set of samples are filtered are substantially identical.
• the hole array filter through which the first set of samples are filtered and the hole array filter through which the second set of samples are filtered are separate hole array filters.
• each samples is filtered through a separate hole array filter.
• Another embodiment of the present invention includes a method of enhancing the identification of peaks in a mass spectrometric method. The method may comprise the steps of filtering a sample through a hole array filter and generating mass spectra data from the sample.
• Yet another embodiment ofthe present invention may include a method of increasing sensitivity and specificity in disease detection.
• the method may comprise -the steps of generating a first set of mass spectra data from a first set of biological fluid samples from a population with disease A after filtration ofthe first set of biological fluid samples through a hole array filter and generating a second set of mass spectra data from a second set of biological fluid samples from a population without disease A after filtration ofthe second set of biological fluid samples through a hole array filter.
• the method may also include the step ofcomparing the first and second sets of mass spectra data, wherein a difference between corresponding peaks in the first and second sets of mass spectra data indicates at least one disease A negative marker.
• the structure ofthe hole array filter through which the first set of samples are filtered and the structure ofthe hole array filter through which the second set of samples are filtered are substantially identical.
• the hole array filter through which the first set of samples are filtered and the hole array filter through which the second set of samples are filtered are separate hole array filters.
• each samples is filtered through a separate hole array filter.
• one embodiment of the present invention may include an apparatus for filtering biological fluid to predict response to disease treatment comprising at least one hole array filter.
• a first set of samples from a population that respond to a treatment of a disease A may be filtered through the at least one hole array filter and a first set of mass spectra data may be generated from the first set of samples after filtering through the at least one hole array filter.
• a second set of samples from a population that does not respond to the same treatment of disease A may also be filtered through the at least one hole array filter and a second set of mass spectra data may be generated from the second set of samples after filtering through the at least one hole array filter.
• the at least one hole array filter includes at least one first hole array filter and at least one second hole array filter, each having substantially identical structure.
• the at least one first hole array " filter may be used for filtering the first set of samples and the at least one second hole array filter may be used for filtering the second set of samples. Each sample may be filtered through a separate hole array filter.
• one embodiment of the present invention may include an apparatus for filtering biological fluid to detect disease by measuring mass spectra data of filtered biological fluid comprising at least one hole array filter.
• a first set of biological fluid samples from a population with disease A may be filtered through the. at least one hole array filter and a first set of mass spectra data may be generated from the first set of biological fluid samples after filtering through the at least one hole array filter.
• a second set of biological fluid samples from a population without disease A may also be •filtered through the at least one hole array filter and a second set of mass spectra data may be generated from the second set of biological fluid samples after filtering through the at least one hole array filter.
• the first and second sets of mass spectra data may be compared, wherein a difference between corresponding peaks in the first and second sets of mass spectra data may indicate at least one disease A negative marker.
• the at least one hole array filter includes at least one first hole array filter and at least one second hole array filter, each having substantially identical structure. The at least one first hole array filter may be used for filtering the first set of samples and the at least one second hole array filter may be used for filtering the second set of samples. Each sample may be filtered through a separate hole array filter.
• one embodiment of the present invention may include an apparatus for filtering biological fluid to enhance the identification of peaks in a mass spectrometric method comprising a hole array filter.
• a biological fluid sample may be filtered through the hole array filter, and mass spectra data may be generated from the filtered biological fluid sample.
• one embodiment of the present invention may include a method for detecting at least one negative marker for detecting a disease.
• the method may comprise steps of generating a first set of mass spectra data from a first set of biological fluid samples from a population with the disease after filtration of the first set of biological fluid samples through a hole array filter, and generating a second set of mass spectra data from a second set of biological fluid samples from a population without the disease after filtration of the second set of biological fluid samples through a hole array filter.
• the method may also comprise a step of comparing the first and second sets of mass spectra data, wherein a difference between corresponding peaks in the first and second sets of mass spectra data indicates at least one negative marker for detecting the disease.
• the structure of the hole array filter through which the first set of samples are filtered and the structure of the hole array filter through which the second set of samples are filtered are substantially identical.
• the hole array filter through which the first set of samples are filtered and the hole array filter through which the second set of samples are filtered are separate hole array filters.
• each sample is filtered through a separate hole array filter.
• one embodiment of the present invention may include a method for detecting a disease in a test subject.
• the method may comprise a step of utilizing the at least one negative marker detected by the method as explained in the preceding paragraph as a diagnostic marker to detect the disease in the test subject.
• the method may comprise steps of filtering a biological fluid sample from the test subject through a hole array filter; and generating mass spectra data from the filtered biological fluid to evaluate an amount(s) of the at least one negative marker.
• the method may also comprise a step of diagnosing the disease in the test subject based on the amount(s).
• Figure 1 is a flow chart illustrating a method according to one embodiment of the present invention.
• Figures 2-1 to 2-11 are chromatograms of normal pre-f ⁇ ltered and post-filtered sera samples.
• Figures 2-12 to 2-19 are chromatograms of pre-f ⁇ ltered and post-filtered sera samples known to have lung cancer.
• Figure 3a shows a cross section of a filter for use in the present invention.
• Figure 3b shows a top view of a hole array of the filter.
• FIGS 4a-4g show the steps that may used in making filters in accordance with the instant invention.
• Figures 5-1 to 5-34 show chromatograms of pre-filtered sera vs. post-filtered sera showing the enhancement of a peak in a chemosensitivity screening assay.
• Figures 6a- 1 to 6a-32 show the chromatograms of pre-filtered sera vs. post- filtered sera of lung cancer patients.
• Figures 6b- 1 to 6b-34 show the chromatograms of pre-filtered sera vs. post- filtered sera of normal patients.
• Figures 7-1 to 7-42 shows the chromatograms of pre-filtered sera vs. post-filtered sera of pancreatic cancer patients.
• Figures 8-1 to 8-12 show the chromatograms of pre-filtered urine vs. post- filtered urine of both normal and bladder cancer patients.
• Figure 9 shows steps that may be used in making filters in accordance with the instant invention.
• Figures 10a to 1Oe illustrate the results of filtering of serum through a hole array filter according to one embodiment of the present invention.
• the present invention comprises, but is not limited to, methods for predicting and detecting diseases, and methods for predicting response to the treatment of diseases.
• the present invention is especially effective for predicting, detecting, and predicting the response to the treatment of diseases such as lung cancer, pancreatic cancer, and bladder cancer, but is in no way limited to those diseases. This is because one of the principles embraced by invention relates to the removal of unwanted substances in the samples which results in better peak generation and cleaner data. As such, the filters and methods of the instant invention are not disease specific.
• Mass spectroscopic chromatograms were first compared to find differences between normal fluid and fluid of humans with a certain disease, identified herein as disease A. While chromatograms are illustrated in the present disclosure, one of ordinary skill in the art will realize that the use of, and analysis of, any plot of frequency versus time may be utilized without deviating from the scope and spirit of the present invention. This may include, but is not limited to, spectrograms.
• the compared chromatograms focused on a high "molecular range because the differences were thought to be not in small molecules but proteins.
• a special peak difference in the high molecular range could not be identified.
• Normal serum chromatograms show high peaks at the spots corresponding to A arid B, but disease A fluid chromatograms do not show any peak or show only small peaks at those spots.
• mass spectroscopic chromatograms were compared between groups responding to a particular chemotherapy treatment with those that did not respond to that particular chemotherapy treatment. A peak was identified in • a substantial number of the non-responders.
• the filters for use in the present invention may comprise an array of holes formed in a silicon membrane of about 3 to 20 ⁇ m in thickness.
• the membrane thickness is between about 6-10 ⁇ m. If the thickness is less than 6 ⁇ m, the hole array area may become very fragile. If the thickness is more than 10 ⁇ m, filtration time may increase due to the resistance of the hole surface area A. Thickness of more than 10- ⁇ m may also increase the difficulty of making smooth holes.
• the size of the hole array may be between 1 mm by 1 mm and 10 mm by 10 mm. If the area is smaller than 1 mm by 1 mm, the amount of filtered biological fluid may not be enough to generate adequate data.
• the size of the holes in the array may be from about 1 ⁇ m to 20 ⁇ m, and preferably about 1- 10 ⁇ m. In this instance, the term "size" refers to diameter for a circular hole and diagonal for a square hole. If the size is smaller than 1 ⁇ m, the filter hole array area may break when negative pressure is applied. If the size is larger than 10 ⁇ m, unwanted compounds of biological fluid may tend to go through the filter holes and the filtration process may become insufficient.
• the hole pitch, or distance between holes may be about three times the size of the holes (preferably 3-30 ⁇ m) but may be more or less than three times the hole size depending on the particular application (see Figs. 3 a and 3b).
• Hole array filters according to the present invention may consist of mainly two areas — a thin area with a hole array, and a thick outer area to improve filter rigidity.
• the material of the filter may be rigid and may be easily processed to precise designed patterns, as one of ordinary skill in the art will realize.
• Filter materials may include, but are not limited to, materials such as metal or semiconductor material.
• One example is Si(thick layer)/SiO2/Si(thin layer with hole array). If Si(thick layer) is tapered toward Si(thin layer with hole array), the flow of biological fluid through the hole array filter becomes smoother.
• Another material that maybe used for hole array filters is Ni/Cu.
• the hole array filter material should be rigid and with evenly made holes matching the designed size.
• the filters used in the present invention may be made by any method known in the art of lithography or filter making.
• a silicon substrate of about 575 ⁇ m thickness may be used as the starting material.
• a thin layer of silicon dioxide may then be formed on one side of the substrate using any common method such as chemical vapor deposition (CVD) 5 or through further oxidation of the surface portion of the substrate by exposure to an oxygen containing plasma.
• the silicon dioxide layer may be about 2 ⁇ m thick.
• a thin layer of silicon may be formed on the oxide layer by any method such as CVD or thin film crystallization (see FIG.4A).
• the substrate may then be flipped over so the thin crystalline silicon film is on the bottom or backside of the substrate. This silicon naturally develops a very thin layer of silicon dioxide of a thickness on the order of a few Angstroms.
• This substrate is typically called Silicon On Isolator (SOI) substrate.
• SOI Silicon On Isolator
• the resist material may be coated on the SOI surface.
• Resist material can be photoresist for photo exposure such as Ultra Violet light and electron beam resist for electron beam exposure at the following processes.
• Patterned mask may then be applied onto, or in proximity to, the resist.
• ionizing radiation such as ultra violet light or electron beam may be applied to the resist through the patterned mask.
• unnecessary pattern portion of the resist may be removed by removing material such as solvent.
• an Si layer may be etched using either a dry or a wet process to make a certain shaped hole array, as shown in FIG. 4B, after removing the whole resist. In such cases, silicon dioxide layer works as etching stopping layer.
• a protective layer may then be applied over the entire substrate including over the hole array (FIG. 4C).
• a portion of the protective layer on the top side of the substrate and symmetrically arranged compared to the underlying hole array but wider than the hole may then be removed through a mask and resist etching process (FIG. 4D).
• a wet etch of the exposed substrate may then be performed until the oxide layer is reached resulting in the exposure of the oxide layer surrounding the underlying hole array and tapered walls of the side of the exposed silicon substrate, as shown in FIG. 4E.
• the remainder of the protective layer may then be removed by a wet or dry etching process as shown in FIG. 4F.
• the exposed portion of the oxide layer may then be removed by a wet or dry etching process resulting in a finished filter as shown in FIG. 4G.
• Filters in accordance with the instant invention may also be made with other materials including, but not limited to, Ni/Cu. As one of ordinary skill in the art will realize, the steps used to form such filters will be similar to those above and shown in FIG. 9.
• any processes may be used to form a hole array in a thin layer of silicon.
• the thicknesses of the different layers and sizes of the holes and distances between the holes are provided as exemplary only and are not meant to be limiting in any manner.
• the word "hole” is not meant to be limited to a void of any particular shape but may be round, square, triangle, or any other shape. As such, cross sectioning of the holes need not be cylindrical in shape.
• the filter material is not limited to silicon as the filter may comprise any common filter material.
• Bio samples include tissue (e.g., from biopsies), blood, serum, plasma, nipple aspirate, urine, tears, saliva, cells, soft and hard tissues, organs, semen, feces, and the like.
• tissue e.g., from biopsies
• blood serum
• plasma e.g., fetal bovine serum
• nipple aspirate
• urine tears, saliva, cells
• soft and hard tissues e.g., feces, and the like.
• the biological samples may be obtained from any suitable organism including eukaryotic, prokaryotic, or viral organisms.
• the biological samples may include biological molecules including macromolecules such as polypeptides, proteins, nucleic acids, enzymes, DNA, RNA, polynucleotides, oligonucleotides, carbohydrates, oligosaccharides, polysaccharides; fragments of biological macromolecules set forth above, such as nucleic acid fragments, peptide fragments, and protein fragments; complexes of biological macromolecules set forth above, such as nucleic acid complexes, protein-DNA complexes, receptor-ligand complexes, enzyme-substrate, enzyme inhibitors, peptide complexes, protein complexes, carbohydrate complexes, and polysaccharide complexes; small biological molecules such as amino acids, nucleotides, nucleosides, sugars, steroids, lipids, metal ions, drugs, hormones, amides, amines, carboxylic acids, vitamins and coenzymes, alcohols, aldehydes, ketones, fatty acids, porphyrins,
• the hole array filters identified in the following table were evaluated for their ability to cleanse sample and thereby improve the sensitivity and specificity of the present methods.
• Serum samples were filtered with each of the above filters. Evaluation by MALDI-TOF-MS found the following trend in cleansing effect: 1-1 IP > 5-lOP > 5-20P > 5-55P > 10-40P > 10-11OP. That is, filter 1-1 IP had the greatest cleansing effect for the samples tested. However, each progressive level of filtration increased the ability to identify certain peaks representing mass ion species as compared to the prior level of filtration. Thus, the mass spectra data for samples filtered with the filters had an improved resolution, or enhanced signal to noise ratio, for potential peaks of interest (e.g., spectral peaks corresponding to the mass to charge ratio of different molecules) compared to unfiltered samples.
• potential peaks of interest e.g., spectral peaks corresponding to the mass to charge ratio of different molecules
• FIGS. 10a to 1Oe Hole array filters having a hole diameter of 2 ⁇ m, 3 ⁇ m, 4 ⁇ m, 5 ⁇ m, 6 ⁇ m, 7 ⁇ m, 8 ⁇ m and 9 ⁇ m were also evaluated for cleansing effect. The results of these experimentations are shown in FIGS. 10a to 1Oe.
• FIG. 10a is a chromatogram from a sample of cancer serum filtered through a hole array filter with a diameter of 2 ⁇ m.
• FIG. 10b is a chromatogram from a sample of cancer serum filtered through a hole array filter with a diameter of 5 ⁇ m.
• FIG. 10c is a chromatogram from a sample of cancer serum filtered through a hole array filter with a diameter of 9 ⁇ m.
• FIG. 10a is a chromatogram from a sample of cancer serum filtered through a hole array filter with a diameter of 2 ⁇ m.
• FIG. 10b is a chromatogram from a sample of cancer serum filtered through a hole array
• 1Od is a chromatogram from samples of cancer serum filtered through hole array filters with diameters of 3 ⁇ m, 4 ⁇ m, 5 ⁇ m, 6 ⁇ m, 7 ⁇ m, and 8 ⁇ m.
• FIG. 1Oe is a chromatogram from samples of normal serum filtered through hole array filters with diameters of 3 ⁇ m, 4 ⁇ m, 5 ⁇ m, 6 ⁇ m, 7 ⁇ m, and 8 ⁇ m.
• the results from the 9 ⁇ m hole filters tend to give less filtering effect than the smaller hole filters. In some cases, this maybe because the larger holes allow an increased amount of those substances (e.g., molecules) in the biological fluid that reduce the signal to noise ratio to pass through the filter. However, in other cases, the larger hole filters may be sufficient to filter out enough of noise-causing substances to increase the resolution of potential peaks of interest. Nonetheless, this evaluation indicated that a filter hole size of between 2 and 9 ⁇ m work well because enough of the noise-causing substances were filtered to increase the relevant peaks in the final data.
• the following examples 1-4 illustrate specific testing and analysis of sera using the methods and apparatus of the present invention. Each example utilizes the method illustrated in FIG. 1. As shown in FIG.
• a biological fluid 1 may be filtered through a filter 2, with the resulting sample being collected and prepared for mass spectroscopy 3.
• the sample may then be loaded onto a mass spectroscopy plate 4, and the samples may be run through a mass spectrometer 5.
• the mass spectrometry data may then be analyzed as discussed in the examples below.
• MALDI-TOF-MS was used to generate a spectra sample data set representing distinct protein/peptide patterns in serum.
• sera either from patients with lung cancer or healthy controls were obtained before surgical procedures. All final diagnoses were confirmed by histopathology and all controls were at high risk for lung cancer, but without evidence of lung cancer based on clinical presentation and CT scan examination.
• the sera were prepared for evaluation by a mass spectrometer by making a matrix of serum samples.
• the mass spectrometer matrix contained saturated alpha- cyano-4-hydroxycinnamic acid in 50% acetonitrile-0.05% trifluoroacetic acid (TFA).
• the sera were diluted 1:1000 in 0.1% n-Octyl ⁇ -D-Glucopyranoside. 0.5 ⁇ L of the matrix was placed on each defined area of a sample plate with 384 defined areas and 0.5 ⁇ L serum from each individual was added to the defined areas followed by air dry. Samples and their locations on the sample plates were recorded for accurate data interpretation.
• An Axima-CFR MALDI-TOF mass spectrometer manufactured by Kratos Analytical Inc. was used. The instrument was set to the following specifications: tuner mode, linear; mass range, 0 to about 5,000; laser power, 90; profile, 100; shots per spot, 5. The output of the mass spectrometer was stored in computer storage in the form of a sample data set.
• the serum was diluted 1:10 in 0.1% n-Octyl ⁇ -D- Glucopyranoside.
• the micro tubes were cut individually from Micro Amp 8 strip tubes. A hole was made in the bottom part of the micro tube by using a needle (Becton Dickinson 20Gl) having a diameter of 0.9 mm or less.
• the micro tube with hole was 5 placed on the metal plate of a gel-pak suction apparatus (air pump) and the hole was adjusted to the same air-flow direction of the air pump.
• the filter was. placed on the top of the micro tube. 20 ⁇ l of serum was loaded on the upper part of filter. The serum solution spread out, filling the inner square of the filter. The negative air flow was applied by pumping the air pump manually.
• FIGS. 2-1 to 2-19 A comparison between normal sera data and lung cancer sera data is illustrated in FIGS. 2-1 to 2-19, with normal pre-filtered and post-filtered samples shown in FIGS. 2-1 to 2-11 and lung cancer ("Disease A") pre-filtered and post-filtered samples shown in FIGS. 2-12 to 2-19.
• FIGS. 6a-l to 6a-32 show chromatograms of pre- filtered sera versus post-filtered sera of lung cancer patients
• FIGS. 6b- 1 to 6b-34 A comparison between normal sera data and lung cancer sera data is illustrated in FIGS. 2-1 to 2-19, with normal pre-filtered and post-filtered samples shown in FIGS. 2-1 to 2-11 and lung cancer ("Disease A") pre-filtered and post-filtered samples shown in FIGS. 2-12 to 2-19.
• FIGS. 6a-l to 6a-32 show chromatograms of pre- filtered sera versus post-filtered sera of lung cancer patients
• FIGS. 6b- 1 to 6b-34 A comparison between normal sera data
• FIGS. 2-1 to 2-19 and 6a-l to 6b-34 show that the use of the filter accentuates the differences between the pre-filter chromatograms and the post-filter chromatograms. This enhancement improves the detection of the peaks.
• the data presented in the above tables and the chromatograms illustrated in FIGS. 2-1 to 2-19 illustrate that filtering of the samples resulted in a ten percent increase in sensitivity at spot A and a nine percent increase in sensitivity at spot B in the normal chormatograms.
• peaks at spot A were shown pre-filtering while all of the normal chromatograms illustrated peaks at spot A after filtering.
• peaks at spot B were shown pre-filtering while seven of the normal chromatograms illustrated peaks at spot B after filtering.
• the data presented in FIGS indicates an increase in specificity in the chromatograms of samples having the disease.
• biological molecules may include, but are not limited to, macromolecules such as polypeptides, proteins, nucleic acids, enzymes, DNA, RNA, polynucleotides, oligonucleotides, carbohydrates, oligosaccharides, polysaccharides, fragments of biological macromolecules (e.g. nucleic acid fragments, peptide fragments, and protein fragments), complexes of biological macromolecules (e.g.
• nucleic acid complexes protein-DNA complexes, receptor-ligand complexes, enzyme- substrate, enzyme inhibitors, peptide complexes, protein complexes, carbohydrate complexes, and polysaccharide complexes
• small biological molecules such as amino acids, nucleotides, nucleosides, sugars, steroids, lipids, metal ions, drugs, hormones, amides, amines, carboxylic acids, vitamins and coenzymes, alcohols, aldehydes, ketones, fatty acids, porphyrins, carotenoids, plant growth regulators, phosphate esters and nucleoside diphospho-sugars, synthetic small molecules such as pharmaceutically or therapeutically effective agents, monomers, peptide analogs, steroid analogs, inhibitors, mutagens, carcinogens, antimitotic drugs, antibiotics, ionophores, antimetabolites, amino acid analogs, antibacterial agents, transport inhibitors, surface- active agents (surfactants), mitochondrial and chloroplast
• pancreatic cancer screening performed using the apparatus and methods according to the present invention discussed above.
• MALDI-TOF-MS was used to generate a spectra sample data set representing distinct protein/peptide patterns in serum.
• fluid either from patients with pancreatic cancer or healthy controls were obtained before surgical procedures. All final diagnoses were confirmed by histopathology and all controls were at high risk for pancreatic cancer, but without evidence of pancreatic cancer based on clinical presentation and CT scan examination.
• the sera were prepared for evaluation by the mass spectrometer by making a matrix of serum samples.
• the mass spectrometer matrix contained saturated alpha- cyano-4-hydroxycinnamic acid in 50% acetonitrile-0.05% trifluoroacetic acid (TFA).
• TFA trifluoroacetic acid
• the fluids were diluted 1:1000 in 0.1% n-Octyl ⁇ -D-Glucopyranoside.
• 0.5 ⁇ L of the matrix was placed on each defined area of a sample plate with 384 defined areas and 0.5 ⁇ L serum from each individual was added to the defined areas followed by air dry. Samples and their locations on the sample plates were recorded for accurate data interpretation.
• An Axima-CFR MALDI-TOF mass spectrometer manufactured by Kratos Analytical I ⁇ c. was used. The instrument was set to the following specifications: tuner mode, linear; mass range, 0 to about 5,000; laser power, 90; profile, 100; shots per spot, 5.
• the serum was diluted 1:10 in 0.1% n-Octyl ⁇ -D- Glucopyranoside.
• the micro tubes were cut individually from Micro Amp 8 strip tubes. A hole was made in the bottom part of the micro tube by using a needle (Becton Dickinson 20Gl) having a diameter of 0.9 mm or less.
• the micro tube with hole was placed on the metal plate of a gel-pak suction apparatus (air pump) and the hole was adjusted to the same air-flow direction of the air pump.
• the filter was placed on the top of the micro tube. 20 ⁇ l of serum was loaded on the upper part of filter. The serum solution spreads out, filling the inner square of filter. The negative air flow was applied by pumping the air pump manually.
• the dropped sera solutions from the filter to the micro tube were collected and transferred to the new tube.
• the filtered serum was further diluted 1:100 in 0.1% n-Octyl ⁇ -D-Glucopyranoside.
• 0.5 ⁇ L, of the matrix was placed on each defined area of a sample plate with 384 defined areas and 0.5 ⁇ L serum from each individual was added to the defined areas followed by air dry. Samples and their locations on the sample plates were recorded for accurate data interpretation.
• An Axima-CFR MALDI-TOF mass spectrometer manufactured by Kratos Analytical Inc. was used. The instrument was set to the following specifications: tuner mode, linear; mass range, 0 to about 5,000; laser power, 90; profile, 100; shots per spot, 5.
• the output of the mass spectrometer was stored in computer storage in the form of a sample data set.
• the use of the filter accentuated the differences between the presence of peaks in. the pre-filter and post-filter chromatograms for pancreatic cancer. These results will be apparent to one of ordinary skill in the art by examining FIGS. 7-1 to 7-42 in the same manner as the examination of the results of the lung cancer screening discussed above. Clearly, the use of the filter enhanced the detection of the relevant peaks.
• pancreatic cancer the present invention is not meant to be limited to any particular disease.
• the present invention is applicable to any disease that may show a difference in mass chromatograms compared to those of normal patients.
• Exemplary diseases may include, but are not limited to, cancers of the respiratory, gastrointestinal, renal, CNS, endocrine and blood systems or any other diseases or disease processes (e.g. necrosis, apoptosis) in which there are potential alterations in molecules contained in biological fluid (e.g. blood and blood derivatives, urine, cerebral spinal fluid, sputum, lavage).
• Such biological molecules may include, but are not limited to, macromolecules such as polypeptides, proteins, nucleic acids, enzymes, DNA, RNA, polynucleotides, oligonucleotides, carbohydrates, oligosaccharides, polysaccharides, fragments of biological macromolecules (e.g. nucleic acid fragments, peptide fragments, and protein fragments), complexes of biological macromolecules (e.g.
• nucleic acid complexes protein-DNA complexes, receptor-ligand complexes, enzyme-substrate, enzyme inhibitors, peptide complexes, protein complexes, carbohydrate complexes, and polysaccharide complexes
• small biological molecules such as amino acids, nucleotides, nucleosides, sugars, steroids, lipids, metal ions, drugs, hormones, amides, amines, carboxylic acids, vitamins and coenzymes, alcohols, aldehydes, ketones, fatty acids, porphyrins, carotenoids, plant growth regulators, phosphate esters and nucleoside diphospho-sugars, synthetic small molecules such as pharmaceutically or therapeutically effective agents, monomers, peptide analogs, steroid analogs, inhibitors, mutagens, carcinogens, antimitotic drugs, antibiotics, ionophores, antimetabolites, amino acid analogs, antibacterial agents, transport inhibitors, surface-active agents (surfactants), mitochondrial and
• MALDI-TOF-MS was used to generate a spectra sample data set representing distinct protein/peptide patterns in urine.
• urine either from patients with bladder cancer or healthy controls were obtained before surgical procedures. All final diagnoses were confirmed by histopathology and all controls were at high risk for bladder cancer, but without evidence of bladder cancer based on clinical presentation and CT scan examination.
• the samples were prepared for evaluation by the mass spectrometer by making a matrix of urine samples.
• the mass spectrometer matrix contained saturated alpha- cyano-4-hydroxycinnamic acid in 50% acetonitrile-0.05% trifluoroacetic acid (TFA).
• the fluids were diluted 1:1000 in 0.1% n-Octyl ⁇ - D-Glucopyranoside. 0.5 ⁇ L of the matrix was placed on each defined area of a sample plate with 384 defined areas and 0.5 ⁇ L urine from each individual was added to the defined areas followed by air dry. Samples and their locations on the sample plates were recorded for accurate data interpretation.
• An Axima-CFR MALDI-TOF mass spectrometer manufactured by Kratos Analytical Inc. was used. The instrument was set to the following specifications: tuner mode, linear; mass range, 0 to about 5,000; laser power, 90; profile, 100; shots per spot, 5. The output of the mass spectrometer was stored in computer storage in the form of a sample data set.
• the urine was diluted 1 :10 in 0.1% n-Octyl ⁇ -D- Glucopyranoside.
• the micro tubes were cut individually from Micro Amp 8 strip tubes. A hole was made in the bottom part of the micro tube by using needle (Becton Dickinson 20Gl) having a diameter of 0.9 mm or less.
• the micro tube with hole was placed on the metal plate of a gel-pak suction apparatus (air pump) and the hole was adjusted to the same air-flow direction of the air pump.
• the filter was placed on the top of the micro tube. 20 ⁇ l of urine was loaded on the upper part of filter. The urine solution spreads out, filling the inner square of filter. The negative air flow was applied by pumping the air pump manually.
• the dropped urine solutions from the filter to the micro tube were collected and to transferred to the new tube.
• the filtered urine was further diluted 1 :100 in 0.1% n-Octyl ⁇ -D-Glucopyranoside.
• 0.5 ⁇ L of the matrix was placed on each defined area of a sample plate with 384 defined areas and 0.5 ⁇ L urine from each individual was added to the defined areas followed by air dry. Samples and their locations on the sample plates were recorded for accurate data interpretation.
• An Axima-CFR MALDI-TOF mass spectrometer manufactured by Kratos Analytical Inc. was used. The instrument was set to the following specifications: tuner mode, linear; mass range, 0 to about 5,000; laser power, 90; profile, 100; shots per spot, 5.
• the output of the mass spectrometer was stored in computer storage in the form of a sample data set.
• the use of the filter accentuated the differences between the presence of peaks in the pre-filter and post-filter chromatograms for bladder cancer.
• FIGS. 8-1 to 8-12 FIGS. 8-1 to 8-6 are chromatograms of samples known to have bladder cancer while FIGS. 8-7 to 8- 12 are normal chromatograms).
• the use of the filter enhanced the detection of the relevant peaks.
• bladder cancer bladder cancer
• the present invention is not meant to be limited to any particular disease.
• the present invention is applicable to any disease that may show a difference in mass chromatograms compared to those of normal patients.
• Exemplary diseases may include, but are not limited to, cancers of the respiratory, gastrointestinal, renal, CNS, endocrine and blood systems or any other diseases or disease processes (e.g. necrosis, apoptosis) in which there are potential alterations in molecules contained in biological fluid (e.g. blood and blood derivatives, urine, cerebral spinal fluid, sputum, lavage).
• Such biological molecules may include, but are not limited to, ⁇ macromolecules such as polypeptides, proteins, nucleic acids, enzymes, DNA, RNA, polynucleotides, oligonucleotides, carbohydrates, oligosaccharides, polysaccharides, fragments of biological macromolecules (e.g. nucleic acid fragments, peptide fragments, and protein fragments), complexes of biological macromolecules (e.g.
• nucleic acid complexes protein-DNA complexes, receptor-ligand complexes, enzyme- substrate, enzyme inhibitors, peptide complexes, protein complexes, carbohydrate complexes, and polysaccharide complexes
• small biological molecules such as amino acids, nucleotides, nucleosides, sugars, steroids, lipids, metal ions, drugs, hormones, amides, amines, carboxylic acids, vitamins and coenzymes, alcohols, aldehydes, ketones, fatty acids, porphyrins, carotenoids, plant growth regulators, phosphate esters and nucleoside diphospho-sugars, synthetic small molecules such as pharmaceutically or therapeutically effective agents, monomers, peptide analogs, steroid analogs, inhibitors, mutagens, carcinogens, antimitotic drugs, antibiotics, ionophores, antimetabolites, amino acid analogs, antibacterial agents, transport inhibitors, surface- active agents (surfactants), mitochondrial and chloroplast
• MALDI- TOF-MS was used to generate a spectra sample data set representing distinct mass over charge ion peaks in serum. These peaks may represent biological molecules include macromolecules such as polypeptides, proteins, nucleic acids, enzymes, DNA, RNA, polynucleotides, oligonucleotides, carbohydrates, oligosaccharides, polysaccharides, fragments of biological macromolecules (e.g. nucleic acid fragments, peptide fragments, and protein fragments), complexes of biological macromolecules (e.g.
• nucleic acid complexes protein-DNA complexes, receptor-ligand complexes, enzyme- substrate, enzyme inhibitors, peptide complexes, protein complexes, carbohydrate complexes, and polysaccharide complexes
• small biological molecules such as amino acids, nucleotides, nucleosides, sugars, steroids, lipids, metal ions, drugs, hormones, amides, amines, carboxylic acids, vitamins and coenzymes, alcohols, aldehydes, ketones, fatty acids, porphyrins, carotenoids, plant growth regulators, phosphate esters and nucleoside diphospho-sugars, synthetic small molecules such as pharmaceutically or therapeutically effective agents, monomers, peptide analogs, steroid analogs, inhibitors, mutagens, carcinogens, antimitotic drugs, antibiotics, ionophores, antimetabolites, amino acid analogs, antibacterial agents, transport inhibitors, surface- active agents (surfactants), mitochondrial and chloroplast
• the sera were prepared for evaluation by the mass spectrometer by making a matrix of serum samples.
• the mass spectrometer matrix contained saturated alpha- cyano-4-hydroxycinnamic acid in 50% acetonitrile-0.05% tri nuoroacetic acid (TFA).
• TFA tri nuoroacetic acid
• the sera were diluted 1:1000 in 0.1% n-Octyl ⁇ -D-Glucopyranoside.
• 0.5 ⁇ L of the matrix was placed on each defined area of a sample plate with 384 defined areas and 0.5 ⁇ L serum from each individual was added to the defined areas followed by air dry. Samples and their locations on the sample plates were recorded for accurate data interpretation.
• the serum was diluted 1 : 10 in 0.1% n-Octyl ⁇ -D- Glucopyranoside.
• the micro tubes were cut individually from Micro Amp 8 strip tubes. A hole was made in the bottom part of the micro tube by using a needle (Becton Dickinson 20Gl) having a diameter of 0.9 mm or less.
• the micro tube with hole was placed on the metal plate of a gel-pak suction apparatus (air pump) and the hole was adjusted to the same air-flow direction of the air pump.
• the filter was placed on the top of the micro tube. 20 ⁇ l of serum was loaded on the upper part of filter. The serum solution spread out, filling the inner square of filter. The negative air flow was applied by pumping the air pump manually.
• the dropped sera solutions from the filter to the micro tube were collected and to transferred to the new tube.
• the filtered serum was further diluted 1:100 in 0.1% n-Octyl ⁇ -D-Glucopyranoside.
• 0.5 pL of the matrix was placed on each defined area of a sample plate with 384 defined areas and 0.5 ⁇ L serum from each individual was added to the defined areas followed by air dry. Samples and their locations on the sample plates were recorded for accurate data interpretation.
• An Axima-CFR MALDI-TOF mass spectrometer manufactured by Kratos Analytical Inc. was used. The instrument was set to the following specifications: tuner mode, linear; mass range, 0 to about 5,000; laser power, 90; profile, 100; shots per spot, 5.
• Figures 5-1 to 5-34 illustrate comparisons of pre-filter and post-filter samples for responders to a treatment and non-responders to a treatment.
• Figures 5-1 to 5-14 are chromatograms comparing pre-filter and post-filter samples of patients showing response to Taxol-based chemotherapy.
• Figures 5-15 to 5-34 are chromatograms comparing pre-filter and post-filter samples of patients showing no response to Taxol- based chemotherapy. Analysis of the chromatograms reveals that a peak is present at a particular point C in a substantial number of the non-responders that is not present in the responders. As shown in the figures, point C corresponds to a mass over charge ratio of 491.
• the presence of a peak at point C illustrates the non-response to Taxol-based chemotherapy. Therefore, it can be predicted, using the methods of the present invention, that patients whose filtered chromatogram shows a peak at point C may be predicted to be a non-responder to Taxol-based chemotherapy.
• Taxol-based chemotherapy is not meant to be limited to any particular disease treatment.
• the present invention may be used to predict response to treatment for a variety of diseases including, but not limited to, treatment for cancers of the respiratory, gastrointestinal, renal, CNS, endocrine and blood systems or any other diseases or disease processes (e.g. necrosis, apoptosis) in which there are potential alterations in molecules contained in biological fluid (e.g. blood and blood derivatives, urine, cerebral spinal fluid, sputum, lavage).
• biological fluid e.g. blood and blood derivatives, urine, cerebral spinal fluid, sputum, lavage.
• Such biological molecules may include, but are not limited to, macromolecules such as polypeptides, proteins, nucleic acids, enzymes, DNA, RNA, polynucleotides, oligonucleotides, carbohydrates, oligosaccharides, polysaccharides, fragments of biological macromolecules (e.g. nucleic acid fragments, peptide fragments, and protein fragments), complexes of biological macromolecules (e.g.
• nucleic acid complexes protein-DNA complexes, receptor-ligand complexes, enzyme-substrate, enzyme inhibitors, peptide complexes, protein complexes, carbohydrate complexes, and polysaccharide complexes
• small biological molecules such as amino acids, nucleotides, nucleosides, sugars, steroids, lipids, metal ions, drugs, hormones, amides, amines, carboxylic acids, vitamins and coenzymes, alcohols, aldehydes, ketones, fatty acids, porphyrins, carotenoids, plant growth regulators, phosphate esters and nucleoside diphospho-sugars, synthetic small molecules such as pharmaceutically or therapeutically effective agents, monomers, peptide analogs, steroid analogs, inhibitors, mutagens, carcinogens, antimitotic drugs, antibiotics, ionophores, antimetabolites, amino acid analogs, antibacterial agents, transport inhibitors, surface-active agents (surfactants), mitochondrial and
• any suitable mixture or combination of the substances mentioned above may also be included in the biological samples.
• the invention has been described with reference to certain exemplary embodiments thereof, those skilled in the art may make various modifications to the described embodiments of the invention without departing from the scope of the invention.
• the terms and descriptions used herein are set forth by way of illustration only and are not meant as limitations.
• the present invention has been described by way of examples, a variety of compositions and methods would practice the inventive concepts described herein.

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