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ARS Home » Pacific West Area » Albany, California » Western Regional Research Center » Produce Safety and Microbiology Research » Research » Publications at this Location » Publication #353750

Research Project: Molecular Identification and Characterization of Bacterial and Viral Pathogens Associated with Foods

Location: Produce Safety and Microbiology Research

Title: Top-down proteomic identification of a novel antibiotic-induced plasmid-encoded factor from Shiga toxin-producing Escherichia coli (STEC) using MALDI-TOF-TOF-MS/MS and post-source decay

item Fagerquist, Clifton - Keith
item Lee, Bertram
item Zaragoza, William
item Yambao, Jaszemyn
item Quinones, Beatriz

Submitted to: Meeting Proceedings
Publication Type: Proceedings
Publication Acceptance Date: 6/10/2018
Publication Date: 7/13/2018
Citation: Fagerquist, C.K., Lee, B.G., Zaragoza, W.J., Yambao, J.C., Quinones, B. 2018. Top-down proteomic identification of a novel antibiotic-induced plasmid-encoded factor from Shiga toxin-producing Escherichia coli (STEC) using MALDI-TOF-TOF-MS/MS and post-source decay. Proceedings for 66th Conference on Mass Spectrometry and Allied Topics, June 3-7, 2018, San Diego, California.

Interpretive Summary:

Technical Abstract: We identified the expression of a novel antibiotic-induced, plasmid-encoded factor in a subset of Shiga toxin-producing Escherichia coli (STEC) O113:H21 strains displaying different levels of pathogenic potential. Six STEC strains (all Shiga toxin 2a subtypes) were cultured overnight on Luria-Bertani agar supplemented with 1200 ng/mL of mitomycin-C. A one microliter loop of cells were harvested from each plate and transferred to Eppendorf tubes containing 300 microliter of water. The tubes were vortexed briefly and centrifuged at 13,000 rpm for 2 minutes. Sample supernatants were analyzed on a MALDI-TOF-TOF mass spectrometer (AB Sciex) in both MS linear mode and MS/MS reflectron-mode with post-source decay (PSD). The full genomes of all STEC O113:H21 strains were examined by using the Illumina Mi-Seq and Pacific Biosciences RS2 next-generation sequencing platforms. We detected in these strains a prominent protein biomarker in MS linear mode with a mass: 7838 +/- 8 Da. This protein gave a relatively uncomplicated MS/MS-PSD spectrum with multiple complementary b/y fragment ion pairs resulting from polypeptide backbone cleavage on the C-terminal side of aspartic acid (D) and glutamic acid (E) residues due to the aspartic acid effect. In addition, the number of complementary b/y fragment ions suggested that protonation sites, i.e. basic residues, were distributed throughout the polypeptide sequence. The complementary b/y fragment ion pairs allowed a second, independent measurement of the mass of the protein by the formula: [(b - H+) + (y - H+)] = protein mass (Da). Using the three most abundant complementary b/y fragment ion pairs, we calculated the average mass of the protein to be 7834.7 +/- 0.8. An iterative search using the protein mass and the mass-to-charge (m/z) of the most abundant fragment ions of the open reading frames of genomes of these strains (including plasmids) revealed a plasmid-encoded factor having an average mass of 7834.1 Da. Manual inspection of the MS/MS spectrum and comparison to in silico fragment ions for this sequence confirmed the correctness of the identification with a total of nine polypeptide backbone cleavage sites caused by D and E residues resulting in nine complementary b/y fragment ion pairs. When this sequence was uploaded to our top-down proteomic software, it resulted in a highly significant identification: P-value = 8.5 x 10-24. Although the function of this plasmid-encoded factor is not known, its induction by antibiotic exposure may suggest a role in stress response in these pathogenic STEC strains.