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FINGERPRINTING OF C. PARVUM BY MATRIX ASSISTED LASER DESORPTION IONIZATION MASS SPECTROMETRY
Citation:
Glassmeyer, S, J A. Shoemaker, F W. Schaefer III, AND D D. Kryak. FINGERPRINTING OF C. PARVUM BY MATRIX ASSISTED LASER DESORPTION IONIZATION MASS SPECTROMETRY. Presented at Society of Environmental Toxicology and Chemistry Annual Meeting, Baltimore, MD, November 11-15, 2001.
Impact/Purpose:
This particular task is comprised of 4 subtasks: 1.) Characterization of Potential Viral Biomarkers by Mass Spectrometry; 2.) Characterization of Parasites by Mass Spectrometric Techniques;
3.) Rapid Discrimination of Bacterial Indicators of Fecal Contamination and Bacterial Pathogens by Mass Spectrometric Techniques; and 4.) Investigation of Aeromonas Virulence Factors Using Mass Spectrometry.
The purpose of this research project is to use mass spectrometric techniques, such as electrospray ionization (ESI), capillary electrophoresis (CE) and matrix assisted laser desorption ionization (MALDI) mass spectrometry, to provide "protein mass fingerprinting" and protein sequencing information for viruses, bacteria and protozoa that cause waterborne disease. These protein mass fingerprinting libraries will be evaluated to determine whether mass spectrometric techniques can identify protein fingerprints related to the infectivity/viability of selected microorganisms and whether they can differentiate between infective / non-infective genus and strains of the selected microorganisms. The characteristic proteins identified by mass spectrometry as markers of infectivity/viability or strain differentiation can then be used to develop more sensitive microbiological drinking water methods.
Description:
The oocysts of Cryptosporidium parvum, an enteric protozoan pathogen, are responsible for the worst microbial waterborne outbreak of gastroenteritis in recent history. The 1993 outbreak in Milwaukee, WI, sickened approximately 403,000 individuals, resulting in the hospitalization of over 4,000, and the death of nearly 100 people. The current assay for identifying C. parvum is labor intensive and time consuming. Matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) is one tool that may eventually help create a faster, easier assay for Cryptosporidium and other microbial pathogens (including other protozoa, bacteria, and/or viruses). Similar gas chromatography/mass spectrometry (GC/MS), MALDI-TOF MS produces a characteristic "fingerprint" of the proteins contained within a sample. By comparing the mass spectral fingerprints of whole, viable oocysts, to oocysts that have been made non-viable (by means such as heating, freezing, exposure to UV light, and ozonation), as well as isolated oocyst walls, the specific (and potentially unique) proteins that compromise the outer wall of the viable oocysts can be determined. Further analysis using MALDI-TOF MS or liquid chromatography/electrospray/mass spectrometry (LC/ESI/MS), can yield the peptide sequence for these specific proteins. This information can be used to develop more sensitive and precise immunological techniques, such as ELISA and IFA, that focus in on these external parasite proteins in drinking water samples.