Detecting the Unseen Enemies - Bio-warfare
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Detecting the Unseen Enemies - Bio- Detecting the Unseen Enemies - Bio- warfare warfare The overall goal of this project is to use mass spectrometry (MS) to develop The overall goal of this project is to use mass spectrometry (MS) to develop rapid, real-time, and facile methods for remote detection of biological rapid, real-time, and facile methods for remote detection of biological warfare agents in the atmosphere . To facilitate these aims, studies are warfare agents in the atmosphere . To facilitate these aims, studies are conducted using simulants of Bacillus anthracis (our model organism and conducted using simulants of Bacillus anthracis (our model organism and the causative agent of anthrax). The major spore coat proteins of the the causative agent of anthrax). The major spore coat proteins of the simulants are isolated and characterized and then analyzed by MS . Results simulants are isolated and characterized and then analyzed by MS . Results would provide unique molecular signatures enabling the identification of would provide unique molecular signatures enabling the identification of similar biological warfare agents in sample aerosol. similar biological warfare agents in sample aerosol. Adebomi Omikunle Adebomi Omikunle Mississippi State Universit Mississippi State Universit y y Joanne Horn Ph.D., Earth & Environmental Sciences Joanne Horn Ph.D., Earth & Environmental Sciences Directorate, Lawrence Livermore National Directorate, Lawrence Livermore National Laboratory, Laboratory, Office of Defense Programs, U.S. Department of Office of Defense Programs, U.S. Department of Energy Energy Results: Results: Discussion: Discussion: Introduction Introduction Methods: Methods: The aim of these studies is to The aim of these studies is to determine whether different determine whether different biological particles yield different biological particles yield different mass spectrometry signatures. If mass spectrometry signatures. If so, this will be the basis for the so, this will be the basis for the development of a spectrometer that development of a spectrometer that will, in real time, detect and will, in real time, detect and distinguish between different distinguish between different organisms. organisms. The analogs of The analogs of Bacillus anthracis Bacillus anthracis used used for this study are: for this study are: Bacillus subtilis, Bacillus subtilis, Bacillus subtilis var. niger, Bacillus cereus, Bacillus subtilis var. niger, Bacillus cereus, and and Bacillus thuringiensis. Bacillus thuringiensis. This study aims to This study aims to isolate and characterize the spore isolate and characterize the spore coat proteins of these simulants coat proteins of these simulants using sodium dodecyl sulfate (SDS)- using sodium dodecyl sulfate (SDS)- Polyacrylamide gel electrophoresis Polyacrylamide gel electrophoresis (PAGE), ion - exchange (PAGE), ion - exchange chromatography, electroelution chromatography, electroelution techniques and mass spectrometry. techniques and mass spectrometry. The organisms were cultured The organisms were cultured separately in 1/4X tryptone-yeast separately in 1/4X tryptone-yeast sporulation media sporulation media 1 1 . The spores . The spores produced were harvested by produced were harvested by centrifugation, and the coat centrifugation, and the coat proteins extracted by incubating proteins extracted by incubating at 37 at 37 ° ° C. for one hour in 50mM C. for one hour in 50mM TRIS, pH 8, 8M urea, 1% SDS, 50mM TRIS, pH 8, 8M urea, 1% SDS, 50mM dithiothreitol. The resulting dithiothreitol. The resulting extract was dialyzed against 50mM extract was dialyzed against 50mM TRIS, pH 8, 1mM phenylmethyl- TRIS, pH 8, 1mM phenylmethyl- sulfonyl fluoride . The crude sulfonyl fluoride . The crude extracts were then either extracts were then either partially purified using gel partially purified using gel elution elution 2 2 or ion-exchange or ion-exchange chromatography on SP sepharose chromatography on SP sepharose using 50mM NaPO using 50mM NaPO 4 4 buffer, pH7.5 and buffer, pH7.5 and a linear gradient of 0 - 1M NaCl. a linear gradient of 0 - 1M NaCl. Results were analyzed using SDS- Results were analyzed using SDS- PAGE PAGE 2 2 . . References: References: 1) 1) Lazzarini, Robert A. and E. Santangelo. Lazzarini, Robert A. and E. Santangelo. 1967. 1967. “Medium-dependent Alteration of “Medium-dependent Alteration of Lysine Transfer Lysine Transfer Ribonucleic Acid in Ribonucleic Acid in Sporulating Sporulating Bacillus subtilis Bacillus subtilis Cells.” Journal Cells.” Journal of Bacteriol. 94: 125-130. of Bacteriol. 94: 125-130. 2) 2) Jacobs, E. and A. Clad. 1986. Jacobs, E. and A. Clad. 1986. “Electroelution of “Electroelution of fixed and stained fixed and stained membrane proteins from membrane proteins from preparative SDS-polyacrylamide gels into preparative SDS-polyacrylamide gels into membrane trap.” Anal. Biochem. 154: membrane trap.” Anal. Biochem. 154: 583-589. 583-589. - - Visualization and molecular Visualization and molecular weight weight analysis of the major analysis of the major coat proteins of coat proteins of the four species the four species of Bacillus was of Bacillus was achieved achieved by SDS-PAGE (fig. 2). by SDS-PAGE (fig. 2). - - Visualization of eluted and Visualization of eluted and partially partially purified 22.5Kd & purified 22.5Kd & 24Kd proteins from 24Kd proteins from B. cereus B. cereus and and B. B. thuringiensis thuringiensis by SDS- by SDS- PAGE PAGE electroelution was electroelution was accomplished (fig.4) accomplished (fig.4) - - A single ion-exchange A single ion-exchange purification purification achieved the achieved the separation of four separation of four Bacillus Bacillus subtilis subtilis spore coat proteins from crude spore coat proteins from crude extracts (fig.3). extracts (fig.3). - - Mass spectrometry signatures of Mass spectrometry signatures of four four types of Bacillus spores types of Bacillus spores illustrates illustrates significant significant differences (fig. 1), differences (fig. 1), demonstrating that mass demonstrating that mass spectrometry spectrometry is a promising tool is a promising tool for identifying for identifying bacteria. bacteria. - - Future additional efforts Future additional efforts include: large include: large scale scale preparation of spores, use of preparation of spores, use of silver staining techniques on gels silver staining techniques on gels fig. 3. Partially fig. 3. Partially Purified Purified B. subtilis B. subtilis Spore Spore Coat Proteins Coat Proteins Ion Exchange:Sepharose, pH 7.5, 0- Ion Exchange:Sepharose, pH 7.5, 0- 0.5M NaCl, 15% SP SDS-PAGE gel 0.5M NaCl, 15% SP SDS-PAGE gel fig. 2. Bacillus Spore fig. 2. Bacillus Spore Coat Proteins-Crude Coat Proteins-Crude Extracts Extracts Extraction of whole Extraction of whole spores spores 15% SDS-PAGE 15% SDS-PAGE Fig. 4. Gel- Fig. 4. Gel- Electroeluted Spore Coat Electroeluted Spore Coat Proteins of Proteins of B. cereus & B. B. cereus & B. thuringiensis thuringiensis Electrophoretic Separation - 15% Electrophoretic Separation - 15% SDS-PAGE (fig. 4) SDS-PAGE (fig. 4) Bacillus Bacillus niger niger Bacillus Bacillus thuringiensis thuringiensis Bacillus Bacillus cereus cereus fig. 1. Time of flight - MS fig. 1. Time of flight - MS Spectra of three different Spectra of three different Bacillus spore species Bacillus spore species B. thuringiensis 22.5Kd B. thuringiensis 24Kd Low MW B. cereus 22.5Kd B. cereus 24Kd Polypept ide UCRL-#####-##-# Work performed under the auspices of the U.S. Department of Energy by the Lawrence Livermore National Laboratory under Contract W-7405-Eng-48.
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Detecting the Unseen Enemies - Bio-warfare. Adebomi Omikunle Mississippi State Universit y Joanne Horn Ph.D., Earth & Environmental Sciences Directorate, Lawrence Livermore National Laboratory, Office of Defense Programs, U.S. Department of Energy. - PowerPoint PPT Presentation
Transcript of Detecting the Unseen Enemies - Bio-warfare
Detecting the Unseen Enemies - Bio-warfareDetecting the Unseen Enemies - Bio-warfare
The overall goal of this project is to use mass spectrometry (MS) to develop The overall goal of this project is to use mass spectrometry (MS) to develop rapid, real-time, and facile methods for remote detection of biological rapid, real-time, and facile methods for remote detection of biological
warfare agents in the atmosphere . To facilitate these aims, studies are warfare agents in the atmosphere . To facilitate these aims, studies are conducted using simulants of Bacillus anthracis (our model organism and conducted using simulants of Bacillus anthracis (our model organism and
the causative agent of anthrax). The major spore coat proteins of the the causative agent of anthrax). The major spore coat proteins of the simulants are isolated and characterized and then analyzed by MS . simulants are isolated and characterized and then analyzed by MS .
Results would provide unique molecular signatures enabling the Results would provide unique molecular signatures enabling the identification of similar biological warfare agents in sample aerosol.identification of similar biological warfare agents in sample aerosol.
Adebomi Omikunle Adebomi Omikunle Mississippi State UniversitMississippi State Universityy
Joanne Horn Ph.D., Earth & Environmental Sciences Joanne Horn Ph.D., Earth & Environmental Sciences
Directorate, Lawrence Livermore National Laboratory,Directorate, Lawrence Livermore National Laboratory,Office of Defense Programs, U.S. Department of EnergyOffice of Defense Programs, U.S. Department of Energy
Results:Results:
Discussion:Discussion:
IntroductionIntroduction Methods:Methods:
The aim of these studies is to determine The aim of these studies is to determine whether different biological particles yield whether different biological particles yield different mass spectrometry signatures. If so, different mass spectrometry signatures. If so, this will be the basis for the development of a this will be the basis for the development of a spectrometer that will, in real time, detect and spectrometer that will, in real time, detect and distinguish between different organisms. distinguish between different organisms.
The analogs of The analogs of Bacillus anthracisBacillus anthracis used for used for this study are: this study are: Bacillus subtilis, Bacillus Bacillus subtilis, Bacillus subtilis var. niger, Bacillus cereus, subtilis var. niger, Bacillus cereus, and and Bacillus thuringiensis. Bacillus thuringiensis. This study aims to This study aims to isolate and characterize the spore coat isolate and characterize the spore coat proteins of these simulants using sodium proteins of these simulants using sodium dodecyl sulfate (SDS)-Polyacrylamide gel dodecyl sulfate (SDS)-Polyacrylamide gel electrophoresis (PAGE), ion - exchange electrophoresis (PAGE), ion - exchange chromatography, electroelution techniques chromatography, electroelution techniques and mass spectrometry. and mass spectrometry.
The organisms were cultured separately in The organisms were cultured separately in 1/4X tryptone-yeast sporulation media1/4X tryptone-yeast sporulation media11. . The spores produced were harvested by The spores produced were harvested by centrifugation, and the coat proteins centrifugation, and the coat proteins extracted by incubating at 37extracted by incubating at 37°°C. for one C. for one hour in 50mM TRIS, pH 8, 8M urea, 1% hour in 50mM TRIS, pH 8, 8M urea, 1% SDS, 50mM dithiothreitol. The resulting SDS, 50mM dithiothreitol. The resulting extract was dialyzed against 50mM TRIS, extract was dialyzed against 50mM TRIS, pH 8, 1mM phenylmethyl-sulfonyl fluoride . pH 8, 1mM phenylmethyl-sulfonyl fluoride . The crude extracts were then either The crude extracts were then either partially purified using gel elutionpartially purified using gel elution22 or ion- or ion-exchange chromatography on SP exchange chromatography on SP sepharose using 50mM NaPOsepharose using 50mM NaPO44 buffer, buffer, pH7.5 and a linear gradient of 0 - 1M NaCl. pH7.5 and a linear gradient of 0 - 1M NaCl. Results were analyzed using SDS-PAGEResults were analyzed using SDS-PAGE22..References:References:1)1) Lazzarini, Robert A. and E. Santangelo. 1967. Lazzarini, Robert A. and E. Santangelo. 1967. “Medium-dependent Alteration of Lysine Transfer “Medium-dependent Alteration of Lysine Transfer Ribonucleic Acid in Sporulating Ribonucleic Acid in Sporulating Bacillus subtilisBacillus subtilis Cells.” Journal of Bacteriol. 94: 125-130.Cells.” Journal of Bacteriol. 94: 125-130.2)2) Jacobs, E. and A. Clad. 1986. “Electroelution of Jacobs, E. and A. Clad. 1986. “Electroelution of fixed and stained membrane proteins from fixed and stained membrane proteins from preparative SDS-polyacrylamide gels into preparative SDS-polyacrylamide gels into membrane trap.” Anal. Biochem. 154: 583-589.membrane trap.” Anal. Biochem. 154: 583-589.
-- Visualization and molecular weight Visualization and molecular weight analysis of the major coat proteins of analysis of the major coat proteins of the four species of Bacillus was the four species of Bacillus was achieved by SDS-PAGE (fig. 2).achieved by SDS-PAGE (fig. 2).-- Visualization of eluted and partially Visualization of eluted and partially purified 22.5Kd & 24Kd proteins from purified 22.5Kd & 24Kd proteins from B. B. cereuscereus and and B. thuringiensisB. thuringiensis by SDS- by SDS- PAGE PAGE electroelution was electroelution was accomplished (fig.4)accomplished (fig.4)-- A single ion-exchange purification A single ion-exchange purification achieved the separation of four achieved the separation of four BacillusBacillus subtilissubtilis spore coat proteins from crude spore coat proteins from crude extracts (fig.3).extracts (fig.3).-- Mass spectrometry signatures of four Mass spectrometry signatures of four types of Bacillus spores illustrates types of Bacillus spores illustrates significant differences (fig. 1), significant differences (fig. 1), demonstrating that mass spectrometry demonstrating that mass spectrometry is is a promising tool for identifying a promising tool for identifying bacteria. bacteria. -- Future additional efforts include: large Future additional efforts include: large scale preparation of spores, use of scale preparation of spores, use of silver staining techniques on gels to silver staining techniques on gels to improve sensitivity, N-terminal analysis improve sensitivity, N-terminal analysis of of the isolated proteins, and simulated the isolated proteins, and simulated weaponization of the analogs.weaponization of the analogs.
fig. 3. Partially Purified fig. 3. Partially Purified B. B. subtilissubtilis Spore Coat Proteins Spore Coat Proteins
Ion Exchange:Sepharose, pH 7.5, 0-0.5M Ion Exchange:Sepharose, pH 7.5, 0-0.5M NaCl, 15% SP SDS-PAGE gelNaCl, 15% SP SDS-PAGE gel
fig. 2. Bacillus Spore Coat fig. 2. Bacillus Spore Coat Proteins-Crude Extracts Proteins-Crude Extracts
Extraction of whole sporesExtraction of whole spores 15% SDS-PAGE15% SDS-PAGE
Fig. 4. Gel-Electroeluted Fig. 4. Gel-Electroeluted Spore Coat Proteins of Spore Coat Proteins of
B. cereus & B. thuringiensisB. cereus & B. thuringiensisElectrophoretic Separation - 15% SDS-PAGE Electrophoretic Separation - 15% SDS-PAGE
(fig. 4)(fig. 4)
Bacillus Bacillus nigerniger
Bacillus Bacillus thuringiensisthuringiensis
Bacillus Bacillus cereuscereus
fig. 1. Time of flight - MS Spectra of fig. 1. Time of flight - MS Spectra of three different Bacillus spore species three different Bacillus spore species
B. thuringiensis 22.5Kd
B. thuringiensis 24Kd
Low MW B. cereus 22.5Kd
B. cereus 24Kd
Polypeptide
UCRL-#####-##-#Work performed under the auspices of the U.S. Department of Energy by the Lawrence Livermore National Laboratory under Contract W-7405-Eng-48.
