|Fagerquist, Clifton - Keith|
Submitted to: Analyst
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/23/2011
Publication Date: 2/21/2011
Citation: Fagerquist, C.K., Sultan, O. 2011. Induction and identification of disulfide-intact and disulfide-reduced beta-subunit of Shiga toxin 2 from Escherichia coli O157:H7 using MALDI-TOF-TOF-MS/MS and top-down proteomics. Analyst.136:1739-1746. DOI: 10.1039/C0AN00909A. Interpretive Summary: Shiga toxin (Stx) is a protein toxin and one of the causes of severe illness, kidney failure or even death as a result of exposure to Escherichia coli O157:H7. Increasingly, Stx has also been found in non-O157 E. coli. Developing rapid methods for the detection and identification of Stx (as well as other bacterial toxins) is a critical element of food safety. We have developed a technique for rapid detection and identification of the beta-subunit of Stx2 from E. coli O157:H7 using mass spectrometry-based top-down proteomics. E. coli O157:H7 was cultured overnight under conditions that induce expression of Stx2. The intact protein toxin is ionized by matrix-assisted laser desorption ionization (MALDI), isolated, fragmented and the fragment ions analyzed by time-of-flight-time-of-flight (TOF-TOF) tandem mass spectrometry (MS/MS). The toxin subunit was identified from its sequence-specific fragment ions using top-down proteomic software developed in-house at the USDA. In addition, MS/MS resulted in a characteristic fragmentation pattern that confirmed the presence of a single disulfide bond in this toxin subunit. Reduction of the disulfide bond improved top-down identification. This rapid proteomic approach to protein toxin identification provides definitive proof of the ability of a bacterial strain to produce a toxin and thus its potential to cause severe illness in humans.
Technical Abstract: The disulfide-intact and disulfide-reduced beta-subunit of Shiga toxin 2 (beta-Stx2) from Escherichia coli O157:H7 (strain EDL933) has been identified by matrix-assisted laser desorption/ionization time-of-flight-time-of-flight tandem mass spectrometry (MALDI-TOF-TOF-MS/MS) and top-down proteomic analysis using software developed in-house. E. coli O157:H7 was induced to express Stx2 by culturing on solid agar media supplemented with 10-50 ng/mL of ciprofloxacin (CP). Bacterial cell lysates at each CP concentration were analyzed by MALDI-TOF-MS. A prominent ion at mass-to-charge (m/z) ' 7820 was observed for the CP concentration range: 10-50 ng/mL, reaching a maximum signal intensity at 20 ng/mL. A complex MS/MS spectrum was obtained of the ion at m/z ' 7820 by post-source dissociation resulting in top-down proteomic identification as the mature, signal peptide-removed, disulfide-intact beta-Stx2. Eight fragment ion triplets (each spaced delta m/z ' 33 apart) were also observed in the MS/MS spectrum, the result of backbone cleavage between the two cysteine residues (that form the intra-molecular disulfide bond) and symmetric and asymmetric cleavage of the disulfide bond. The middle fragment ion of each triplet, from symmetric disulfide bond cleavage, was matched to an in silico fragment ion formed from cleavage of the backbone at a site adjacent to an aspartic acid or glutamic acid residue. The flanking fragment ions of each triplet, from asymmetric disulfide bond cleavage, were not matched because their corresponding in silico fragment ions are not represented in the database. An easier to interpret MS/MS spectrum was obtained for the disulfide-reduced beta-Stx2 which resulted in an improved top-down identification.