# Presentation: 808462

## Detecting the Unseen Enemies - Bio-warfare

**Detecting the Unseen Enemies - Bio-warfare**

***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  warfare agents in the atmosphere . To facilitate  these  aims, studies are conducted using simulants of Bacillus anthracis (our  model organism and the causative agent of anthrax).  The  major spore coat proteins of the simulants are  isolated and  characterized  and then analyzed by MS .  Results would provide unique molecular signatures enabling the identification of  similar biological warfare agents in sample aerosol.***

**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**

**Results:**

**Discussion:**

**Introduction**

**Methods:**

**	****The aim of these studies is to determine whether different biological particles yield different mass spectrometry signatures.  If so, this will be the basis for the development of a spectrometer that will, in real time, detect and distinguish between different organisms.  **
**	****The analogs of *****Bacillus anthracis***** used for this study are: *****Bacillus subtilis, Bacillus subtilis var. niger, Bacillus cereus, *****and *****Bacillus thuringiensis.  *****This study aims to isolate and characterize the spore coat proteins of these simulants  using sodium dodecyl sulfate (SDS)-Polyacrylamide gel electrophoresis (PAGE), ion - exchange chromatography, electroelution techniques and mass spectrometry. **

**The organisms were cultured separately in 1/4X tryptone-yeast sporulation media****1****.  The spores produced were harvested by centrifugation, and the coat proteins extracted by incubating at 37****°****C. for one hour in  50mM TRIS, pH 8, 8M urea, 1% SDS, 50mM dithiothreitol.  The resulting extract was dialyzed against 50mM TRIS, pH 8, 1mM phenylmethyl-sulfonyl fluoride .  The crude extracts were then either partially purified using gel elution****2**** or ion-exchange chromatography on SP sepharose using 50mM NaPO****4**** buffer, pH7.5 and a linear gradient of 0 - 1M NaCl. Results were analyzed using SDS-PAGE****2****.**
**References:**
**1)****	****Lazzarini, Robert A. and E. Santangelo.  1967.  ****	****	****“Medium-dependent Alteration of Lysine Transfer ****	****Ribonucleic Acid in Sporulating *****Bacillus subtilis***** ****	****Cells.”  Journal of Bacteriol.  94: 125-130.**
**2)****	****Jacobs, E. and A. Clad.  1986.  “Electroelution of ****	****fixed and stained membrane proteins from ****	****	****	****preparative SDS-polyacrylamide gels into ****	****	****	****membrane trap.”  Anal. Biochem.  154: 583-589.**

**-****	****Visualization and molecular weight ****	****	****analysis of  the major coat proteins of ****	****the four species of Bacillus was ****	****	****	****achieved by SDS-PAGE (fig. 2).**
**-****	****Visualization of eluted and partially ****	****	****purified 22.5Kd & 24Kd proteins from ****	*****B. cereus***** and *****B. thuringiensis***** by SDS-****	****PAGE electroelution was  ****	****	****	****	****	****accomplished (fig.4)**
**-****	****A single ion-exchange purification ****	****	****achieved the separation of four *****Bacillus***** ****	*****subtilis***** spore coat proteins from crude ****	****extracts (fig.3).**
**-****	****Mass spectrometry signatures of four ****	****types of Bacillus spores illustrates ****	****	****significant differences (fig. 1), ****	****	****	****demonstrating that mass spectrometry ****	****is a promising tool for identifying ****	****	****	****bacteria. **
**-****	****Future additional efforts include: large ****	****scale preparation of spores, use of ****	****	****silver staining techniques on gels to ****	****	****improve sensitivity, N-terminal analysis ****	****of the isolated proteins, and simulated ****	****weaponization of the analogs.**

**fig. 3. Partially Purified *****B. subtilis***** Spore Coat Proteins   **
**Ion Exchange:Sepharose, pH 7.5, 0-0.5M NaCl, 15% SP SDS-PAGE gel**

**fig. 2. Bacillus Spore Coat Proteins-Crude Extracts **
**Extraction of whole spores**
** ****15% SDS-PAGE**

**Fig. 4.  Gel-Electroeluted Spore Coat Proteins of **
**B. cereus & B. thuringiensis**
**Electrophoretic Separation - 15% SDS-PAGE (fig. 4)**

***Bacillus niger***

***Bacillus thuringiensis***

***Bacillus cereus***

**fig. 1.  Time of flight - MS Spectra of 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.