title: Top-down proteomic identification of furin-cleaved α-subunit of Shiga toxin 2 from Escherichia coli O157:H7 using MALDI-TOF-TOF-MS/MS.
abstract: A method has been developed to identify the α-subunit of Shiga toxin 2 (α-Stx2) from Escherichia coli O157:H7 using matrix-assisted laser desorption/ionization time-of-flight-time-of-flight tandem mass spectrometry (MALDI-TOF-TOF-MS/MS) and top-down proteomics using web-based software developed in-house. Expression of Stx2 was induced by culturing E. coli O157:H7 on solid agar supplemented with an antibiotic that elicits the bacterial SOS-response. Bacterial cell lysates were incubated in the presence of furin, a human enzyme, that cleaves α-Stx2 into A1 (~28 kDa) and A2 (~5 kDa) protein fragments. A subsequent disulfide reduction step unlinked A1 from A2. MALDI-TOF-MS of the furin-digested/disulfide-reduced sample showed a peak at mass-to-charge (m/z) 5286 that corresponded to the A2 fragment. No peak was observed that corresponded to the A1 fragment although its presence was confirmed by bottom-up proteomics. The peak at m/z 5286 was definitively identified by MALDI-TOF-TOF-MS/MS and top-down proteomics as the A2 fragment of α-Stx2.
authors.affiliation: Western Regional Research Center, Agricultural Research Service, U.S. Department of Agriculture, 800 Buchanan Street, Albany, CA 94710, USA. clifton.fagerquist@ars.usda.gov
authors.forename: Clifton K
authors.initials: CK
authors.lastname: Fagerquist
authors.forename: Omar
authors.initials: O
authors.lastname: Sultan
mesh_terms: D000595:Amino Acid Sequence; D019453:Escherichia coli O157 / Q000737:chemistry* / Q000378:metabolism; D045683:Furin / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D021122:Protein Subunits; D040901:Proteomics / Q000379:methods*; D020539:Sequence Analysis, Protein; D022641:Shiga Toxin 2 / Q000737:chemistry* / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D053719:Tandem Mass Spectrometry / Q000379:methods
chemical_list: D021122:Protein Subunits; D022641:Shiga Toxin 2; D045683:Furin