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THE APPLICATION OF MASS SPECTROMETRY TO THE STUDY OF MICROORGANISMS
Citation:
Shoemaker, J A. AND S Glassmeyer. THE APPLICATION OF MASS SPECTROMETRY TO THE STUDY OF MICROORGANISMS. Presented at U.S. EPA's Research on Micoorganisms in Drinking Water Workshop, Cincinnati, OH, August 5-7, 2003.
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 purpose of this research project is to use state-of-the-art mass spectrometric techniques, such as electrospray ionization (ESI) and matrix assisted laser desorption ionization (MALDI) mass spectrometry (MS), to provide "protein mass fingerprinting" and protein sequencing information for microorgansims listed on the 1998 Contaminant Candidate List (CCL) that cause waterborne disease. The responsibility of characterizing and investigating microorganisms has traditionally fallen to microbiologists, but recent advances in mass spectrometry have allowed analytical chemists to also enter the realm of microorganisms.
Protein mass fingerprinting libraries will be developed and evaluated to determine whether MS techniques can identify protein fingerprints related to the infectivity/viability of selected microorganisms and whether they can differentiate between species and strains of selected microorganisms. Sequence information for proteins which are found to be specific or unique to species/strain and infectivity/viability can also be obtained with these MS techniques.
This global proteomic project has a number of subtasks for which preliminary results have been obtained on microorganisms such as coxsackievirus, Cryptosporidium parvum, and enterococci. Through the use of mass spectrometry, a potential viral biomarker of coxsackievirus has been identified which may indicate whether the virus is infectious. A unique mass spectral peak was observed in an infectious coxsackievirus, but was not observed in a non-infectious coxsackievirus. This unique peak may be responsible for viral infectivity, thus, be a potential biomarker.
In addition to viruses, initial experiments were performed to determine the ability of MALDI to analyze C. parvum both in an intact form, as well as oocysts that have been rendered nonviable. MALDI analysis was performed on several different harvests of the intact oocysts, as well as the separated cell walls and sporozoites that make up the oocysts. The analysis of the oocysts walls was inconclusive due to lack of discernable mass spectral peaks, but MALDI analysis of the sporozoites yielded reproducible mass spectra.
Whole enterococci cell protein profiles were evaluated using MALDI as a tool to identify seven different enterococci species. Many mass spectral peaks were shared among the different enterococci species, however, each species showed unique peaks, primarily in the 6,000 to 7,000 m/z region. When environmental isolates were tested, the signature peaks were observed in many of the different isolates, suggesting that these peaks could be used for species identification. Sequence analysis of the environmental isolates by 16S rDNA confirmed the identity of the strains tested, and matched the MALDI identity prediction in 75 % of the samples. The results from this study indicate that the analysis of whole enterococci cells by MALDI generate unique protein profiles which can be used for the rapid identification of fecal enterococci environmental isolates.
Although mass spectrometry currently is not sensitive enough to detect single cells in drinking water, the basic proteomic information obtained with these mass spectrometric techniques can be used to develop more sensitive and precise microbiological techniques that focus on these unique proteins in drinking water samples. These conventional microbiological methods can then be used to gather the occurrence data that will be used to create better EPA regulations for protecting humans from microbiological contaminants in U.S. drinking water supplies.