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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 #382897
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 plasmid and host proteins produced by pathogenic Escherichia coli using MALDI-TOF-TOF tandem mass spectrometry

Author
item Dodd, Claire
item Fagerquist, Clifton - Keith

Submitted to: World Microbe Forum
Publication Type: Abstract Only
Publication Acceptance Date: 4/14/2021
Publication Date: 6/20/2021
Citation: Dodd, C.E., Fagerquist, C.K. 2021. Top-down proteomic identification of plasmid and host proteins produced by pathogenic Escherichia coli using MALDI-TOF-TOF tandem mass spectrometry [abstract]. World Microbe Forum. Available: https://www.abstractsonline.com/pp8/#!/9286/presentation/11668.

Interpretive Summary:

Technical Abstract: Background. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) is becoming a popular technique for the rapid identification of microorganisms. Top-down proteomic analysis using MALDI-TOF-TOF tandem mass spectrometry (MS/MS) goes a step further, enabling rapid identification of specific microbial proteins. We have used the latter technique to identify host and plasmid proteins produced by three Shiga toxin-producing Escherichia coli (STEC) strains. Of particular interest was the identification of plasmid-encoded antibacterial immunity proteins. Methods. Three STEC strains (E. coli O113:H21 strains RM7806 and RM7807 and E. coli O104:H4 (2011 German outbreak strain)) were cultured overnight on LB agar supplemented with either mitomycin-C or ciprofloxacin. A small sample of cells was transferred to a microcentrifuge tube containing 300 microliters of water, vortexed and centrifuged at 13,000 rpm for two minutes. Sample supernatant (0.75 microliter) was spotted onto a stainless steel MALDI target and allowed to dry before being overlayed with sinapinic acid. Data was collected using a 4800 MALDI-TOF-TOF mass spectrometer. Proteins were identified from their mass and prominent fragment ions using top-down proteomic software developed in-house. Results. Five proteins were identified from E. coli O113:H21 strain RM7806: the immunity proteins for the antibacterial colicin E3 (Im3) and bacteriocin (ImBac), cold-shock proteins CspC and CsbD and the phosphocarrier protein: HPr. The N-terminal methionine was post-translationally removed in the mature CspC and Im3. Five proteins were identified from E. coli O113:H21 strain RM7807: Im3, ImBac, cold-shock proteins CspC and CspE, and HPr. The N-terminal methionine was removed in the mature CspC, CspE and Im3. Four proteins were identified from E. coli O104:H4 strain RM14735: YahO and YbgS (homeobox protein), a genomically-encoded hypothetical protein (C22711_3545) and a plasmid-encoded hypothetical protein (O3K_26197). All four proteins had N-terminal signal peptides that were post-translationally removed in the mature protein. Conclusion. MALDI-TOF-TOF-MS/MS and top-down proteomics enable rapid identification of bacterial proteins, allowing for a more detailed analyses beyond the genus and species information obtained by MALDI-TOF-MS.