Induction and identification of disulfide-intact and disulfide-reduced β-subunit of Shiga toxin 2 from Escherichia coli O157:H7 using MALDI-TOF-TOF-MS/MS and top-down proteomics

摘要

The disulfide-intact and disulfide-reduced b-subunit of Shiga toxin 2 (b-Stx2) from Escherichia colinO157:H7 (strain EDL933) has been identified by matrix-assisted laser desorption/ionization time-offlight-ntime-of-flight tandem mass spectrometry (MALDI-TOF-TOF-MS/MS) and top-downnproteomic analysis using software developed in-house. E. coli O157:H7 was induced to express Stx2 bynculturing on solid agar media supplemented with 10–50 ng mLu00021 of ciprofloxacin (CP). Bacterial cellnlysates at each CP concentration were analyzed by MALDI-TOF-MS. A prominent ion at mass-tochargen(m/z) u00037820 was observed for the CP concentration range: 10–50 ng mLu00021, reaching a maximumnsignal intensity at 20 ng mLu00021. Complex MS/MS data were obtained of the ion at m/z u00037820 by postsourcendecay resulting in top-down proteomic identification as the mature, signal peptide-removed,ndisulfide-intact b-Stx2. Eight fragment ion triplets (each spaced Dm/z u000333 apart) were also observednresulting from backbone cleavage between the two cysteine residues (that form the intra-molecularndisulfide bond) and symmetric and asymmetric cleavage of the disulfide bond. The middle fragment ionnof each triplet, from symmetric disulfide bond cleavage, was matched to an in silico fragment ionnformed from cleavage of the backbone at a site adjacent to an aspartic acid or glutamic acid residue.nThe flanking fragment ions of each triplet, from asymmetric disulfide bond cleavage, were not matchednbecause their corresponding in silico fragment ions are not represented in the database. Easier toninterpret MS/MS data were obtained for the disulfide-reduced b-Stx2 which resulted in an improvedntop-down identification.