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Paper to be Presented

Microorganism Biomarker Structure Analysis Using
AP-MALDI Ion Trap Mass Spectrometry

Nelli I. Taranenko^a, Olga V. Protchenko^b and Vladimir M. Doroshenko^a

^a MassTech Inc., 6992 Columbia Gateway Drive, Columbia, MD 21046, USA
^b NIH/NIDDK, 10 Center Drive, Bethesda, MD. 20892-1800

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 for microbial analysis we used Escherichia coli (E.coli) and Saccharomyces cerevisiae as model organisms. These cells were chosen due their ubiquitous natural distribution, relatively small genome size, and lack of pathogenicity. 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.

A simple procedure was used for bacterial protein release and digestion. The samples were treated with immobilized trypsin beads (Poroszyme Bulk Immobilized Trypsin, Applied Biosystems, Foster City, CA). E. coli samples (1 mg/mL) were digested using immobilized trypsin at 65^oC for 15 minutes. The digested peptide mixture was then purified by Millipore C18ZipTips® (Billerica, MA). S. cerevisiae cells were first treated with Zymolyase (ZymoResearch, Orange, CA) at 37^o C for 5 minutes and then subjected to immobilized trypsin using the same protocol as for E. coli cells. 1 µ L of 5 mg/mL α-cyano-4-hydrocinnimic 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 APMALDI-PDF Model 122 ion source (MassTech, Columbia, MD) using positive ionization mode. This work was accomplished using PDF mode of operation.

The results presented will confirm 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. This demonstrates that AP-MALDI can play an important role in fast yet reliable detection/identification of environmental bacteria.