WO2001092872A2 - Methods for using mass spectrometry to identify and classify filamentous fungi, yeasts, molds and pollen - Google Patents
Methods for using mass spectrometry to identify and classify filamentous fungi, yeasts, molds and pollen Download PDFInfo
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- WO2001092872A2 WO2001092872A2 PCT/US2001/016696 US0116696W WO0192872A2 WO 2001092872 A2 WO2001092872 A2 WO 2001092872A2 US 0116696 W US0116696 W US 0116696W WO 0192872 A2 WO0192872 A2 WO 0192872A2
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- 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
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
- C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
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- 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
- G01N33/6851—Methods of protein analysis involving laser desorption ionisation mass spectrometry
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- H—ELECTRICITY
- H01—ELECTRIC ELEMENTS
- H01J—ELECTRIC DISCHARGE TUBES OR DISCHARGE LAMPS
- H01J49/00—Particle spectrometers or separator tubes
- H01J49/02—Details
- H01J49/04—Arrangements for introducing or extracting samples to be analysed, e.g. vacuum locks; Arrangements for external adjustment of electron- or ion-optical components
Definitions
- the present disclosure relates generally to the identification and classification of filamentous fungi, yeasts, molds, toxins produced by fungi, and pollen in environmental and biological samples. More particularly, the present disclosure is directed to a method for the identification and classification of filamentous fungi, yeasts, molds, toxins produced by fungi, and pollen with genus, species and strain specific biomarkers generated by mass spectrometry.
- Mass spectrometers have been employed in identifying such microbes.
- Mass spectrometers are commercially available and can include, for example, single or multiple quadrupole, single or multiple magnetic sector, Fourier Transform ion cyclotron resonance (FTICR), ion trap, and combinations thereof (e.g., ion-trap/time-of-flight).
- FTICR Fourier Transform ion cyclotron resonance
- mass spectrometry is an analytical technique used for the accurate determination of molecular weights, the identification of chemical structures, the determination of the compositions of mixtures, and qualitative and/or quantitative elemental analysis.
- a mass spectrometer generates ions of sample molecules under investigation, separates the ions according to their mass-to-charge ratio (m/z), and measures the relative abundance of each ion. This analysis of the mass distribution of the molecule and its ion fragments can lead to a molecular "fingerprint" for identification.
- TOF mass spectrometer which separates ions according to their mass-to-charge ratio by measuring the time it takes generated ions to travel to a detector. See, e.g., U.S. Patent Nos. 5,045,694 and 5,160,840, the contents of which are incorporated herein by reference.
- TOF mass spectrometers are advantageous because they are relatively simple instruments with virtually unlimited mass-to- charge ratio range. TOF mass spectrometers have potentially higher sensitivity than traditional scanning instruments because they can record all the ions generated from each ionization event. TOF mass spectrometers are particularly useful for measuring the mass-to- charge ratio of large organic molecules where conventional magnetic field mass spectrometers lack sensitivity.
- TOF mass spectrometers typically include an ionization source for generating ions of sample material under investigation.
- the ionization source contains one or more electrodes or electrostatic lenses for accelerating and properly directing the ion beam.
- the electrodes are grids.
- a detector is positioned at a predetermined distance from the final grid for detecting ions as a function of time.
- a drift region exists between the final grid and the detector. The drift region allows the ions to travel, in free flight, a predetermined distance before they impact the detector.
- the flight time of an ion accelerated by a given electric potential is proportional to its mass-to-charge ratio.
- the time-of-flight of an ion is a function of its mass-to-charge ratio, and is approximately proportional to the square root of the mass-to- charge ratio. Assuming the presence of only singly charged ions, the lightest group of ions reaches the detector first and is followed by groups of successively heavier mass groups. In practice, however, ions of equal mass and charge do not arrive at the detector at exactly the same time. This occurs primarily because of the initial temporal, spatial, and kinetic energy distributions of generated ions. These initial distributions lead to broadening of the mass spectral peaks thus limiting the resolving power of TOF spectrometers. The ion source plays a major role in obtaining distinct identifiable peaks.
- Electron Ionization (El).
- the sample is vaporized within the mass spectrometer prior to passing into an electron ionization region, where it is subjected to an electron beam.
- El is limited in its application to small molecules below the range of common bioorganic compounds, (see, e.g., Siuzdak, Mass Spectrometry for Biotechnology, pp. 6-7 (1996).)
- El is inadequate in treating compounds above a molecular weight of about 400 Da because the thermal treatment causes decomposition of the analyte to be tested prior to vaporization and can lead, in many cases, to excessive fragmentation.
- LD low density polyethylene
- FAB fast atom bombardment
- ESI electrospray ionization
- FAB the sample to be analyzed is added to a matrix, usually a nonvolatile solvent in which the sample is dissolved, prior to analysis.
- FAB typically uses a direct insertion probe for sample introduction and a high-energy beam of Xe atoms, Cs + ions, or
- NH4 "1" clusters to sputter the sample and matrix from the probe surface.
- the matrix replenishes the surface with new sample as the ion beam bombards the surface and absorbs most of the incident energy thus minimizing damage to the sample from the high-energy particle beam.
- the matrix is also believed to facilitate the ionization process.
- the two most common matrices used are -nitrobenzyl alcohol and glycerol.
- the ion beam desorbs the sample of interest from the matrix solution into the gas phase; charged molecules are then electrostatically propelled into the mass analyzer.
- Massive Cluster Impact (MCI) a form of FAB known to those skilled in the art, produces multiply charged ions making it more suitable for use with high-molecular weight biopolymers.
- ESI is used to produce gaseous ionized molecules from a liquid solution containing the analyte.
- a fine spray of highly charged droplets is created in the presence of a strong electric field at the tip of a metal nozzle maintained at about 4000 V, which are then attracted to the mass spectrometer inlet. Dry gas, heat, or both are applied to the droplets before they enter the mass spectrometer so that the solvent evaporates from the surface.
- the electric field density on the surface of the droplet increases as its size decreases, eventually leading to the expulsion of ions, which are directed to an orifice leading to the mass analyzer.
- ESI is conducive to the formation of multiply charged molecules, making it possible to obtain spectra from high molecular weight compounds.
- ion generation may continue for some time after the laser pulse terminates causing loss of resolution due to temporal uncertainty.
- the main requirement, and perhaps the main drawback, of LD/MS is that analytes must absorb at the wavelength emitted by the laser. These requirements limit the range of compounds studied using LD/MS.
- the performance of LD may be substantially improved by the addition of a small organic matrix molecule to the sample material.
- This technique known as matrix- assisted laser desorption/ionization (MALDI)
- MALDI matrix- assisted laser desorption/ionization
- MALDI matrix- assisted laser desorption/ionization
- MALDI matrix- assisted laser desorption/ionization
- the molecular ions and/or fragment ions formed at the probe tip are accelerated by the electric field toward a detector through a flight tube, which is a long ( 4 m) field free drift region. Since all molecular ions receive the same amount of energy, the time required for ions to travel the length of the flight tube is dependent on their mass. Thus, low-mass ions have a shorter time of flight (TOF) than heavier ions. All the ions that reach the detector as the result of a single laser pulse produce a transient TOF signal.
- TOF time of flight
- the mass of an unknown analyte is determined by comparing its experimentally determined TOF to TOF signals obtained with ions of known mass.
- the MALDI-TOF-MS technique is capable of determining the mass of proteins of between 1 and 40 kDa with a typical accuracy of ⁇ 0.1%, and a somewhat lower accuracy for proteins of molecular mass above 40 kDa.
- MALDI-TOF-MS has been used for the analysis of high molecular weight biomolecules (1 to 150 kDa) and synthetic polymers and to detect biological warfare agents in environmental air samples. Recent efforts have focused on the identification of bacteria using MALDI-TOF-MS to identify bacteria at the genus, species and strain levels.
- bioorganic compounds in the environment such as, for example, eukaryotic organisms (e.g., filamentous fungi, yeasts and molds), components of these organisms (e.g., spores and toxins produced by fungi) and pollen that contain molecules similar to those found in bacteria that could give rise to a false reading or confusion when analyzing a sample for the presence of bacteria.
- eukaryotic organisms e.g., filamentous fungi, yeasts and molds
- components of these organisms e.g., spores and toxins produced by fungi
- pollen that contain molecules similar to those found in bacteria that could give rise to a false reading or confusion when analyzing a sample for the presence of bacteria.
- Previous techniques for identifying and classifying these bioorganic compounds include, for example, morphological examination of microscopic structures and biochemical tests. A problem associated with these techniques is that an improper preparation of the sample in the laboratory could lead to an inaccurate, if not impossible, identification of the organism.
- Typical air samples collected from the environment are complex mixtures and may contain bacterial cells and spores, fungal spores and fragments, pollen grains, toxins produced by fungi, and other bioorganic compounds.
- threat agents such as bacteria
- bioorganic compounds in the background environment to reduce false positives in a risk assessment situation.
- one analyzing a sample for the presence of biological warfare agents such as, for example, bacteria, bacterial spores and toxins
- a method is provided for generating biomarkers for filamentous fungi, yeasts, molds, toxins produced by fungi, and pollen at the genus, species or strain level employing mass spectrometry. It is also an objective to provide such a method that uses a MALDI-TOF mass spectrometer to generate these biomarkers.
- an unknown sample may be analyzed by mass spectrometry and spectra obtained for that sample may be compared with the foregoing biomarkers to quickly and easily identify the unknown sample.
- the present disclosure thus permits one to more quickly and easily identify and classify these bioorganic compounds than prior art methods.
- biomarker refers to an ion or charged molecular fragment produced by mass spectrometry that produces a unique peak and/or peaks on a mass spectrum at the genus, species and strain level.
- bioorganic compounds refers to whole cells or cellular components of filamentous fungi, yeasts, molds or toxins of fungi, and pollen grains that will generate biomarkers when the cells or cellular components are subjected to mass spectrometry.
- FIG. 1 is a picture of pollen grains of Ambrosia trifida (Giant Ragweed) taken through a Scanning Electron Microscope (SEM).
- FIG. 2 are spectra obtained after MALDI-TOF-MS analysis on Juglans nigra in 4-HCCA, sinapinic acid (0.1% TFA), ferulic acid, and sinapinic acid (5% TFA) matrices.
- FIG. 3 are spectra obtained after MALDI-TOF-MS analysis on Kochia scoparia in 4-HCCA, sinapinic acid (0.1% TFA), ferulic acid, and sinapinic acid (5% TFA) matrices.
- FIG. 4 are spectra obtained after MALDI-TOF-MS analysis on Ambrosia trifida in 4-HCCA, sinapinic acid (0.1% TFA), ferulic acid, and sinapinic acid (5% TFA) matrices.
- FIG. 5 are spectra obtained after MALDI-TOF-MS analysis on Populus deltiodes in 4-HCCA, sinapinic acid (0.1% TFA), ferulic acid, and sinapinic acid (5% TFA) matrices.
- FIG. 6 are spectra obtained after MALDI-TOF-MS analysis on Populus nigra italica in 4-HCCA, sinapinic acid (0.1% TFA), ferulic acid, and sinapinic acid (5% TFA) matrices.
- FIG. 7 are a group of spectra obtained after MALDI-TOF-MS analysis on pollen grains in ferulic acid matrix: the spectra are for (from bottom to top) Juglans nigra,
- FIG. 8 are spectra obtained after MALDI-TOF-MS analysis on Juglans nigra and bacterial environmental isolates (Staphylococcus sp., Micrococcus sp., and Bacillus sp.) in sinapinic acid.
- bioorganic compounds selected from the group consisting of filamentous fungi, yeasts, molds, toxins produced by fungi, and pollen grains at the genus, species and strain levels employing mass spectrometry, and the use of these compounds to generate individual biomarkers based on components of these compounds, are described hereinbelow. Accordingly, a library of the biomarkers may advantageously be constructed to aid in future identification of filamentous fungi, yeasts, molds, toxins produced by fungi, and pollen and, in turn, used in conjunction with other libraries of organisms such as, for example, bacteria, to accurately characterize and identify a pure sample or the components of a mixed sample.
- the library can also be employed to characterize and identify components of a sample in a laboratory which, in turn, can be used in conjunction with other instruments or techniques, e.g., microscopy, PCR, biochemical testing via manual or automated systems, immunoassays, etc., to identify the bioorganic compounds.
- instruments or techniques e.g., microscopy, PCR, biochemical testing via manual or automated systems, immunoassays, etc.
- filamentous fungi for analyzing herein are eukaryotic organisms with multicellular structures bounded by a rigid cell wall containing, for example, chitin.
- the fungal cell wall is a structure that is subject to change and modification at different stages in the life of a fungus.
- the wall is ordinarily composed of at least a skeletal or micro fibrillar component located on the inner side of the wall and usually embedded in an amorphous matrix material that extends to the outer surface of the wall.
- the skeletal component consists of at least some highly crystalline, water-insoluble materials that include, for example, beta- linked glucans and chitin, while the matrix consists mainly of polysaccharides, e.g., alpha- glucans, glycoproteins, etc., that are mostly water soluble. Additional components that may be present in the cell walls of fungi include, but are not limited to, lipids, melanins, D- galactosamine polymers, polyuronids, cellulose, sterol, ergosterol, etc.
- Fungi will ordinarily grow as multicellular colonies, e.g., mushrooms or molds, and form a mycelium through a mass of branching, interlocking filaments, or hyphae. Although these branches may be interrupted by cross-walls, the passage of cytoplasm between compartments is possible. Both sexual and asexual reproduction can occur in fungi. In asexual reproduction, spores, known as "conidia", are borne externally at the tips of budding projections formed at various locations along the filaments. Most fungal spores range in size, e.g., from about 2 to about 50 ⁇ m. Spores and hyphal fragments of fungi are ubiquitous in air where they are sometimes the major pollutant and sources of infection or allergic reactions.
- the families represented by the filamentous fungi herein include, but are not limited to, Phycomycetes, Ascomycetes, e.g., Neurospora, Aspergillus and Penicillium, Basidiomycetes and Deuteromycetes.
- filamentous fungi may be a cell of a species of, but not limited to, Acremonium spp., Alternaria spp., Arthrinium spp., Aureobasidium spp., Beauveria spp., Bipolaris spp., Borytis spp., Chaetomium spp., Chrysonilia spp., Cladosoporium spp., Cunninghamella spp., Curvularia spp., Drechslera spp., Emmonsia spp., Epiccoccum spp., Fusarium spp., Humicola spp., Microsporum spp., Mucor spp., Myceliophthora spp., Paecilomyces spp., Pithomyces spp., Rhizomucor spp., Rhizopus spp., Scopulariops
- Yeasts and molds are known to those skilled in the art to be members of the fungal kingdom.
- Yeast for use herein are ordinarily a simple form of fungi that generally consist of single cells rather than hyphae. They typically reproduce asexually by budding (e.g., a small outgrowth on the cell's surface which increases in size until a wall forms to separate the new individual from the parent) and fission.
- Some yeast such as, for example, Saccharomyces cerevisiae, (also referred to as brewer's yeast or baker's yeast) can exhibit, under certain conditions, a filamentous mold-like form. These filamentous cells, often referred to as pseudohyphal cells, have an elongated morphology.
- certain fungi, including molds produce toxins such as T-2 mycotoxins and Aflatoxin Bj. In some instances, these toxins can be airborne.
- Pollen herein are ordinarily in the form of grains which are microspores of seed plants containing, for example, a male gametophyte. Some pollen grains are dispersed by the wind and may be allergenic to pollen-sensitive individuals.
- Common pollen grains include those produced by grasses, trees, shrubs and weeds, and include species of, among others, Sorghum spp., Secale spp., Poa spp., Cynodon spp., Dactylis spp., Agrostis spp., Zea spp., Ulmus spp., Juglans spp., Populus spp., Juniperus spp., Fraxinus spp., Betula spp., Alnus spp., Acer spp., Kochia spp., Iva spp., Artemisia spp., and Ambrosia spp. Pollen can also include, for example, proteins, polypeptides, polysaccharides, glycoproteins, and lipoproteins in both the pollen outer wall and cytoplasm.
- the foregoing filamentous fungi, yeasts, molds, toxins produced by fungi, and pollen can be analyzed in the form of, for example, an air sample, lab sample, etc., employing known commercially available mass spectrometers.
- Suitable mass spectrometers for use herein include, but are not limited to, linear or non-linear reflectron time-of-flight, single or multiple quadrupole, single or multiple magnetic sector, Fourier Transform ion cyclotron resonance (FTICR), ion trap, and combinations thereof (e.g., ion-trap/time-of-flight).
- the analysis using mass spectrometry subjects the sample to an ionization source employing commercially available ionization formats which include, but are not limited to, laser deso ⁇ tion methods (including MALDI), FAB, plasma desorption, continuous or pulsed ESI and related methods (e.g., Ionspray or Thermospray), or MCI to produce charged molecular ions. These ions are then propelled into a mass analyzer to obtain a mass spectra.
- MALDI-TOF-MS is the mass spectrometer utilized to classify and identify the bioorganic compounds.
- a matrix solution is first prepared for addition to an unknown sample.
- the matrix advantageously transfers energy nondestructively from the laser beam to the sample, as discussed hereinbelow, thereby producing intact, large molecular ions in the gas phase.
- the matrix is ordinarily formed by combining, for example, a suitable organic acid with an aqueous solvent to form the matrix solution to which the unknown sample that may contain one or more of the foregoing bioorganic compounds is added.
- Suitable organic acids for use herein include, but are not limited to, low molecular weight aromatic organic acids such as, for example, 2,5-dihydroxybenzoic acid, ⁇ -cyano-4-hydroxycinnamic acid (4- HCCA), 3,5-dimethoxy-4-hydroxycinnamic acid (sinapinic acid), tr ⁇ «,s-4-hydroxy-3- methoxycinnamic acid (ferulic acid) and the like and mixtures thereof.
- low molecular weight aromatic organic acids such as, for example, 2,5-dihydroxybenzoic acid, ⁇ -cyano-4-hydroxycinnamic acid (4- HCCA), 3,5-dimethoxy-4-hydroxycinnamic acid (sinapinic acid), tr ⁇ «,s-4-hydroxy-3- methoxycinnamic acid (ferulic acid) and the like and mixtures thereof.
- Suitable solvents for use herein in forming the matrix solution include, but are not limited to, organic solvents such as nitrites, e.g., acetonitrile; alcohols such as mono- or polyfunctional, unsubstituted or substituted, aliphatic alcohols, e.g., lower alkanols such as methanol, ethanol, propanol, etc.; ethers such as tetrahydrofuran, dioxane and diethyl ether; and the like; water and the like and mixtures thereof.
- organic solvents such as nitrites, e.g., acetonitrile
- alcohols such as mono- or polyfunctional, unsubstituted or substituted, aliphatic alcohols, e.g., lower alkanols such as methanol, ethanol, propanol, etc.
- ethers such as tetrahydrofuran, dioxane and diethyl ether; and
- the matrix solution will be formed by adding the organic acid and solvent in various ratios, including, for example, ranging from about 70/30 (v/v) to about 30/70 (v/v) or in a ratio of 50/50 (v/v).
- additional components can be added to the matrix solution to assist in ionization of the analyte such as, e.g., trifluoroacetic acid (TFA). Amounts of these components can vary, ranging from about 0.1% to about 5 % or more.
- the unknown sample which may contain one or more of the foregoing bioorganic compounds is placed on a metal probe, mixed with the foregoing matrix solution and allowed to dry by techniques known in the art, e.g., air- dried at room temperature, placed in a vacuum, etc.
- the unknown sample is ordinarily placed on the probe as a solution and mixed with an excess amount of prepared matrix solution.
- the order in which the sample and matrix solution are placed on the probe and mixed can vary. For example, the sample can first be placed on the probe, matrix solution added and mixed with the sample and then allowed to dry, or the matrix solution can be placed on the probe, sample solution added and mixed with the matrix solution and then allowed to dry.
- a portion of the matrix solution can be placed on the probe, then the sample solution can be added, followed by the additional of the remaining portion of the matrix solution and the mixture then allowed to dry.
- the sample/matrix mixture is then subjected to MS analysis in a MALDI-TOF instrument equipped with at least a nitrogen laser, e.g., the analyte in matrix solution is subjected to irradiation with a pulsed UV or IR laser beam in a vacuum chamber.
- the laser shot produces desorption and ionization of matrix and analyte with resulting charged molecular ions.
- a complete spectrum can be acquired with one laser shot, however, it is more advantageous to obtain and average several hundred laser shots.
- the present disclosure provides at least a method for generating unique mass spectra for filamentous fungi, yeasts, molds, toxins produced by fungi, and pollen grains.
- MALDI matrices used for this study were purchased from Aldrich (Milwaukee, WI, USA) and included 3,5-dimethoxy-4-hydroxycinnamic acid (sinapinic acid), ⁇ -cyano-4-hydroxycinnamic acid (4-HCCA), and trans-4-hydroxy-3- methoxycinnamic acid (ferulic acid). All matrices were supplied and used without further purification. The matrix solutions employed in this study are set forth below in Table 1.
- Calibration standards were purchased from Sigma (St. Louis, MO, USA) and included Cytochrome C and Angiotensin II. Calibration standard solutions were prepared in water with 0.1% TFA.
- MALDI-TOF mass spectra were obtained using a Kratos Kompact MALDI IV mass spectrometer (Manchester, UK) in linear, positive ion mode. A 337nm nitrogen laser was used with 50 laser shots averaged over a single well of a Kratos 20- well slide. [0053] External mass calibrations were performed using Cytochrome C and
- the figures one for each individual pollen species, show qualitatively similar spectral patterns in the mass range between 1 to 12 kD with little spectral variation observed between the different matrix solvent combinations.
- the minor variations e.g., slight mass shifts
- the minor variations may be due to sample-to-sample differences in surface mo ⁇ hology (sample thickness, roughness, homogeneity, etc.) or mo ⁇ hology of the pollen grain itself (pollen grains range in size from 5 to 200 ⁇ m).
- Figure 7 presents all five of the pollen species in a single matrix, ferulic acid.
- MALDI-TOF-MS was run on three bacteria (Staphylococcus sp., Micrococcus sp., and Bacillus sp.) and pollen grains of Juglans nigra.
- MALDI-TOF-MS was performed as described above in Example 1.
- Figure 8 presents the spectra of the three bacteria and the pollen grains of Juglans nigra. From this figure, it can be seen that pollen produces a mass spectral fmge ⁇ rint that can be easily distinguished from the finge ⁇ rints of the bacteria.
- fungi were transferred to Potato Dextrose Agar for culture at room temperature for 5-7 days.
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EP01939338A EP1294923A2 (en) | 2000-05-31 | 2001-05-23 | Methods for using mass spectrometry to identify and classify filamentous fungi, yeasts, molds and pollen |
AU2001264867A AU2001264867A1 (en) | 2000-05-31 | 2001-05-23 | Methods for using mass spectrometry to identify and classify filamentous fungi, yeasts, molds and pollen |
CA002415224A CA2415224A1 (en) | 2000-05-31 | 2001-05-23 | Methods for using mass spectrometry to identify and classify filamentous fungi, yeasts, molds and pollen |
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EP1942194A1 (en) * | 2007-01-08 | 2008-07-09 | Université René Descartes | Method for identifying a germ isolated from a clinical sample |
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WO2015054468A1 (en) * | 2013-10-09 | 2015-04-16 | University Of Maryland, Baltimore | Methods for identifying fungi |
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2001
- 2001-05-23 CA CA002415224A patent/CA2415224A1/en not_active Abandoned
- 2001-05-23 EP EP01939338A patent/EP1294923A2/en not_active Withdrawn
- 2001-05-23 WO PCT/US2001/016696 patent/WO2001092872A2/en not_active Application Discontinuation
- 2001-05-23 AU AU2001264867A patent/AU2001264867A1/en not_active Abandoned
Non-Patent Citations (6)
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AMIRI-ELIASI B ET AL: "Characterization of protein biomarkers desorbed by MALDI from whole fungal cells." ANALYTICAL CHEMISTRY, (2001 NOV 1) 73 (21) 5228-31., - November 2001 (2001-11) XP002187835 * |
DELUCA S J ET AL: "Pyrolysis-mass spectrometry methodology applied to southeast Asian environmental samples for differentiating digested and undigested pollens." ANALYTICAL CHEMISTRY. UNITED STATES OCT 1986, vol. 58, no. 12, October 1986 (1986-10), pages 2439-2442, XP002203274 ISSN: 0003-2700 * |
LI TZU-YING ET AL: "Characterization of Aspergillus spores by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry." RAPID COMMUNICATIONS IN MASS SPECTROMETRY, vol. 14, no. 24, 23 November 2000 (2000-11-23), pages 2393-2400, XP001052923 ISSN: 0951-4198 * |
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WELHAM K J ET AL: "Characterization of fungal spores by laser desorption/ionization time-of-flight mass spectrometry." RAPID COMMUNICATIONS IN MASS SPECTROMETRY., vol. 14, no. 5, 29 February 2000 (2000-02-29), pages 307-310, XP001052924 ISSN: 0951-4198 * |
WELHAM K J ET AL: "Matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry and its application to the analysis of fungal spores." PHARMACY AND PHARMACOLOGY COMMUNICATIONS, vol. 6, no. 3, March 2000 (2000-03), pages 107-111, XP001052985 ISSN: 1460-8081 * |
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Also Published As
Publication number | Publication date |
---|---|
EP1294923A2 (en) | 2003-03-26 |
WO2001092872A3 (en) | 2003-01-30 |
CA2415224A1 (en) | 2001-12-06 |
AU2001264867A1 (en) | 2001-12-11 |
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