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United States Department of Agriculture

Agricultural Research Service

Research Project: Immunodiagnostics to Detect Prions and Other Important Animal Pathogens

Location: Produce Safety and Microbiology Research

Title: Using mass spectrometry to detect and discriminate among Shiga-like toxins in the attomole range.

Authors
item Silva, Christopher
item Erickson-Beltran, Melissa
item Skinner, Craig
item Dynin, Irina
item Hui, Colleen
item Patfield, Stephanie
item He, Xiaohua

Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: April 2, 2014
Publication Date: June 16, 2014
Citation: Silva, C.J., Erickson-Beltran, M.L., Skinner, C.B., Dynin, I.A., Hui, C., Patfield, S.A., He, X. 2014. Using mass spectrometry to detect and discriminate among Shiga-like toxins in the attomole range [abstract]. Meeting Abstract. Innovations in Mass Spectrometry. Paper No. 355.

Technical Abstract: Shiga toxin producing Escherichia coli (STEC) are the cause of a number of serious foodborne illnesses. Much of the virulence associated with these pathogens results from the phage-mediated release of potent Shiga toxins. These toxins are composed of a pentamer of identical subunits (B subunit), which binds the toxin to specific glycolipids on the surface of mammalian cells, and a catalytic subunit (A subunit) that is responsible for the toxic effects. The toxin is subsequently endocytosed into the cell where the A subunit is enzymatically activated, which results in the catalytic inactivation of the cell’s ribosomes. The production of the Shiga toxins is controlled by lambdoid phages ('24B) that infect the E. coli host. Under non-stress conditions the phage’s DNA replicates when the infected bacterium replicates (lysogenically). If the bacterium is stressed, then the phage may reproduce lytically, which results in the production of Shiga toxins, complete phages and the death of the bacterium. An E. coli host may be infected by several phages and not all of them will be viable. Thus the presence of a toxin gene does not necessarily mean that the toxin can be expressed. We developed a multiple reaction monitoring method based on analyzing conserved peptides, derived from the tryptic digestion of the B subunits. Stable-isotope-labeled analogs were prepared and used as internal standards to identify and quantify these characteristic peptides. We determined that this approach can be used to detect, quantify and distinguish among subtypes of Shiga toxins in the low attomole range. In addition we determined that the procedures necessary to perform our mass spectrometry-based analysis completely inactivate the toxins present in the sample. This is a safe and effective method of detecting and quantitating Shiga toxins.

Last Modified: 12/22/2014
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