title: Noncovalent Shiga-like toxin assemblies: characterization by means of mass spectrometry and tandem mass spectrometry.
abstract: Shiga-like toxin 1 (SLTx), produced by enterohemorrhagic strains of Escherichia coli (EHEC), belongs to a family of structurally and functionally related AB(5) protein toxins that are associated with human disease. EHEC infection often gives rise to hemolytic colitis, while toxin-induced kidney damage is one of the major causes of hemolytic uremic syndrome (HUS) and acute renal failure in children. As such, an understanding and analysis of the noncovalent interactions that maintain the quaternary structure of this toxin are fundamentally important since such interactions have significant biochemical and medical implications. This paper reports on the analysis of the noncovalent homopentameric complex of Shiga-like toxin B chain (SLTx-B(5)) using electrospray ionization (ESI) triple-quadrupole (QqQ) mass spectrometry (MS) and tandem mass spectrometry (MS/MS) and the analysis of the noncovalent hexameric holotoxin (SLTx-AB(5)) using ESI time-of-flight (TOF) MS. The triple-quadrupole analysis revealed highly charged monomer ions dissociate from the multiprotein complex to form dimer, trimer, and tetramer product ions, which were also seen to further dissociate. The ESI-TOFMS analysis of SLTx-AB(5) revealed the complex remained intact and was observed in the gas phase over a range of pHs. Theses findings demonstrate that the gas-phase structure observed for both the holotoxin and the isoloated B chains correlates well with the structures reported to exist in the solution phase for these proteins. Such analysis provides a rapid screening technique for assessing the noncovalent structure of this family of proteins and other structurally related toxins.
authors.affiliation: Department of Biological Sciences, University of Warwick, Gibbet Hill Road, Coventry, UK. j.p.williams@warwick.ac.uk
authors.forename: Jonathan P
authors.initials: JP
authors.lastname: Williams
authors.forename: Brian N
authors.initials: BN
authors.lastname: Green
authors.forename: Daniel C
authors.initials: DC
authors.lastname: Smith
authors.forename: Keith R
authors.initials: KR
authors.lastname: Jennings
authors.forename: Katherine A H
authors.initials: KA
authors.lastname: Moore
authors.forename: Susan E
authors.initials: SE
authors.lastname: Slade
authors.forename: Lynne M
authors.initials: LM
authors.lastname: Roberts
authors.forename: James H
authors.initials: JH
authors.lastname: Scrivens
mesh_terms: D006863:Hydrogen-Ion Concentration; D044367:Phase Transition; D011499:Protein Processing, Post-Translational; D021122:Protein Subunits / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D022622:Shiga Toxin 1 / Q000737:chemistry* / Q000378:metabolism*; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods
chemical_list: D021122:Protein Subunits; D011994:Recombinant Proteins; D022622:Shiga Toxin 1