Effect of Environmental Conditions on Identification of Clostridium botulinum Neurotoxin Type A Using AP-MALDI Mass Spectromotry

• AP-MALDI analysis & identification of toxins in the environment
  
  
• Immobilized trypsin treated toxins & simulants
  
  
• Database searching (MascotTM search engine)

The purpose of this study is to develop a rapid and cost-effective technique for the identification of toxins which can be used in forensic science and counterterrorism. Here, we devised a method, which consists of a combination of simple steps: in-situ trypsin digestion of the toxins, purification of peptide mixture and atmospheric pressure (AP) matrix-assisted laser desorption/ionization (MALDI) MS and MS/MS analysis followed by database search identification. The toxins selected were Clostridium botulinum Neurotoxin A and Esherichia coli Shiga-like Toxin 1 and a toxin simulant, ovalbumin.

AP-MALDI experiments were carried out on a ThermoFinnigan ion trap mass spectrometer. BoNT-A was obtained from Metabiologics, Inc. (Madison, WI). The Shiga-like toxin sample was kindly provided by the Division of Gastroenterology and Hepatology at Johns Hopkins University (Baltimore, MD). Chicken ovalbumin, trypsin inhibitors and all chemicals were obtained from Sigma (St. Louis, MO) and urban particulate matter came from NIST (Gaithersburg, MD). All toxins and simulant were digested without reduction and alkylation using immobilized trypsin beads (Poroszyme Bulk Immobilized Trypsin, Applied Biosystems, Foster City, CA) at 65oC for 5 min while shaking. The toxins were purified using Millipore C18 ZipTips® (Bedford, MA). 1 µL of 5 mg/mL α-cyano-4-hydrocinnimic acid in MeOH:AcN:H2O (1:1:1, v/v/v) 0,1% formic acid were overlaid on the samples and allowed to air dry. Experiments were carried on a LCQ Deca XP ion trap MS integrated with an AP-MALDI ion source [1-6] using positive ionization mode. The sensitivity for all analyzed sample was more than adequate; this work was accomplished using the AP-MALDI-PDF Model 122 [7].

Mascot™ (www.matrixscience.com) MS/MS Ion Search was used to confirm the identities of BoNT-A & ovalbumin. The effect of the environment, as represented in trypsin inhibitors (interferent) or debris (particulate matter), was not significant using immobilized trypsin, C-18 clean-up, AP-MALDI and MS/MS analysis. This technique can be applied for rapid identification of biological threats whether they be toxins, as is demonstrated here, vegetative bacteria, spores or viruses.

1. Doroshenko, V., Laiko, V., Taranenko, N., Berkout, V., and Lee H. Int. J. Mass Spectrom. 2002; 221: 39-58.
2. Moyer, S., Cotter, R., and Woods, A. J. Am. Soc. Mass Spectrom. 2002; 13: 274-283.
3. Mehl, J., Cummings, J., Rohde, E. and Yates, N. Rapid Commun. Mass Spectrom. 2003; 17: 1600-1610.
4. Laiko, V., Baldwin, M., and Burlingame, A. Anal. Chem. 2000; 72: 652-657.
5. Madonna, A., Voorhees, K., Taranenko, N., Laiko, V., and Doroshenko, V. Anal. Chem. 2003; 75: 1628-1637.
6. Pribil, P.A.; Patton, E.; Black, G.; Doroshenko, V. and Fenselau, C. J. Mass Spectrom. 2005; 40: 464-474.
7. Tan PV, Laiko VV, Doroshenko VM. Anal Chem. 76, 2004: 2462-2469.