Rapid Identification of Environmental Bacteria Using Atmospheric Pressure MALDI Ion Trap Mass Spectrometry

Nelli I. Taranenko¹, Gavin E. Black¹, Robert M. Serino² and Vladimir M. Doroshenko¹
¹

, Columbia, MD, (www.apmaldi.com), ²Science & Engineering Services, Inc., Columbia, MD

OVERVIEW

AP-MALDI analysis and identification of environmental microorganisms
Immobilized trypsin enzymatically treated spores, vegetative and yeast
Publicly available database searching (MascotTM search engine)

INTRODUCTION

Identification of microbial contamination has potential applications in the pharmaceutical and environmental monitoring. To demonstrate the capabilities of an atmospheric pressure (AP) MALDI interfaced to an ion trap tandem mass spectrometer (ITMS) for microbial analysis we used Bacillus subtilis subsp. subtilis spores (B.ss spores), vegetative B. cereus (B.c veg), Escherichia coli (E.coli) and Saccharomyces cerevisiae as model organisms. We employed a publicly available database, Mascot™ (Matrix Science). These cells were chosen due their ubiquitous natural distribution, relatively small genome size, lack of pathogenicity and ease of laboratory growth. In addition, most fungi & yeast (S. cerevisiae) are typically highly glycosylated. Carbohydrates are still problematic in the development of biomarkers for the identifications of organisms using mass spectrometry analysis. Our continuing goal is to develop a rapid protocol for identification of environmental bacteria based on the recognition of biomarkers by AP-MALDI.

METHODS

The bacterial cells used were B.ss spores and B.c veg (K. Fox, USC, Columbia, MD) & E. coli Subcloning Efficiency™ DH5&alpha™ Chemicallly competent (Invitrogen, Carlsbad, CA). S. cerevisiae and all chemicals including -cyano-4-hydroxycinnamic acid were purchased from Sigma (St. Louis, MO). A simple procedure was used for bacterial protein release and digestion. The B.ss spore samples were treated with immobilized trypsin beads (Poroszyme Bulk Immobilized Trypsin, Applied Biosystems, Foster City, CA) and the vegetative cells were treated with isopropanol and air dried before immobilized trypsin addition.

RESULTS

B.ss spores

B.c veg

E.coli

S.cerevisiae

CONCLUSIONS

Our data confirmed the usefulness of AP-MALDI MS and MS/MS analysis for bacteria identification and will serve to develop and implement a new methodology for identifying different species in the microbial world. Based on this data it is demonstrated that AP-MALDI can play an important role in fast yet reliable detection/identification of environmental bacteria.

REFERENCES

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.

ACKNOWLEDGMENTS:

Many thanks to Karen Fox, Ph.D., University of South Carolina Medical School, Columbia, SC, 29208 and Olga Protchenko, Ph.D., NIDDK, NIH, Bethesda, MD, 20892