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

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. Method name: Top-down of Shiga toxin, Keywords: Top-down proteomics, Mass spectrometry, STEC, Shiga toxin, MALDI-TOF-TOF