Skip to main content
ARS Home » Pacific West Area » Albany, California » Western Regional Research Center » Produce Safety and Microbiology Research » Research » Publications at this Location » Publication #359330

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

Location: Produce Safety and Microbiology Research

Title: Top-down and middle-down proteomic analysis of Shiga toxin using MALDI-TOF-TOF mass spectrometry

item Fagerquist, Clifton - Keith
item Zaragoza, William

Submitted to: MethodsX
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 4/10/2019
Publication Date: 4/16/2019
Citation: Fagerquist, C.K., Zaragoza, W.J. 2019. Top-down and middle-down proteomic analysis of Shiga toxin using MALDI-TOF-TOF mass spectrometry. MethodsX. 6(2019)815-826.

Interpretive Summary: Methods are needed to rapidly identify and characterize pathogenic bacteria. MALDI-TOF mass spectrometry is increasingly utilized for taxonomic identification of bacteria (genus, species, etc.) with at least two platforms now commercially available. However, there is an increasing need to characterize potentially pathogenic bacteria beyond simple taxonomic classification. Although whole genome sequencing (WGS) provides a detailed characterization of the genomic content of a bacterial strain, it does not indicate whether, to what extent or under what conditions are genes expressed. Mass spectrometry-based proteomics can answer specific questions about gene expression by direct measurement of the protein itself. As an analytical technique, the power of mass spectrometry is its high chemical specificity. However, proteomic technologies are still labor-intensive and time-consuming, in part, because the end-point analysis requires sample preparation that is compatible with efficient transfer and ionization of the target protein (or its peptides) into the gas phase. MALDI-TOF-TOF instruments provide the speed of MALDI-TOF with the ability to perform tandem mass spectrometry (MS/MS) allowing identification of specific biomolecules. A method was developed to rapidly detect and identify the expression of Stx from putative STEC strains using antibiotic induction, MALDI-TOF-TOF mass spectrometry and top-down/middle-down proteomics. The method is sensitive and highly specific in the identification of types and subtypes of Stx.

Technical Abstract: The method describes a step-by-step process for analysis of putative Shiga toxin-producing Escherichia coli (STEC) for expression of Shiga toxin (Stx). The technique utilizes antibiotic induction, mass spectrometry and top-down/middle-down proteomic analysis. Stx expression is induced by overnight culturing of a STEC strain on Luria-Bertani agar (LBA) supplemented with DNA-damaging antibiotics. Culturing on agar media avoids sample contamination from salts, small molecules, peptides, etc. present in broth media that would interfere with protein ionization by matrix-assisted laser desorption/ionization (MALDI). No mechanical lysis of bacterial cells is required to release the toxin as the antibiotic triggers the lytic cycle of the bacteriophage resulting in toxin expression and bacterial cell lysis. Unfractionated samples are analyzed by MALDI-time-of-flight-time-of-flight (MALDI-TOF-TOF) mass spectrometry and tandem mass spectrometry (MS/MS) using post-source decay (PSD). New features of the method are the following. Each putative STEC strain is systematically screened for toxin expression using two different antibiotics at two different concentrations: ciprofloxacin at 10 and 20'ng mL-1 and mitomycin-C at 800 and 1200'ng mL-1 to determine the optimal antibiotic and concentration for toxin expression for each strain. The grid-to-source voltage of MALDI-TOF-TOF is optimized to maximize PSD efficiency.