title: Characterization of heat-labile toxin-subunit B from Escherichia coli by liquid chromatography-electrospray ionization-mass spectrometry and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.
abstract: The possibilities of characterizing the heat-labile enterotoxin (LT) of enterotoxigenic Escherichia coli (ETEC) by liquid chromatography electrospray mass spectrometry (LC/ESI-MS) and matrix-assisted laser desorption with time-of-flight mass spectrometry (MALDI-TOF-MS) were investigated. The B subunit from recombinant E. coli (expression in Pichia pastoris) can be detected by LC/ESI-MS expressed in P. pastoris and the charge envelope signals can be observed; LC/ESI-MS and MALDI-TOF-MS analysis allowed the acquisition of labile toxin subunit B (LTB) molecular weight and preliminary structural characterization of LTB toxin. MALDI-TOF analysis after reduction and alkylation of the protein evidenced the presence of one disulfide bond in the structure of the protein. Confirmatory analysis was carried out by detection of most of the tryptic fragments of the B subunit by MALDI-TOF-MS, obtaining total coverage of the protein sequence. Possible biovariations in the toxin can mostly be determined by sequencing, where an increase of molecular mass in the N-terminal side of the protein was identified. This modification may be due to an O-GlcNAc-1-phosphorylation.
authors.affiliation: Department of Preventive Medicine and Public Health, Faculty of Pharmacy, University of Valencia, Av. Vicent Andrés Estellés s/n, 46100 Burjassot, Spain. m.isabel.sospedra@uv.es
authors.forename: I
authors.initials: I
authors.lastname: Sospedra
authors.forename: C
authors.initials: C
authors.lastname: De Simone
authors.forename: J M
authors.initials: JM
authors.lastname: Soriano
authors.forename: J
authors.initials: J
authors.lastname: Mañes
authors.forename: P
authors.initials: P
authors.lastname: Ferranti
authors.forename: A
authors.initials: A
authors.lastname: Ritieni
mesh_terms: D000595:Amino Acid Sequence; D001427:Bacterial Toxins / Q000032:analysis / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002853:Chromatography, Liquid / Q000379:methods*; D004220:Disulfides / Q000737:chemistry; D004768:Enterotoxins / Q000032:analysis / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000032:analysis / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010766:Phosphorylation; D010843:Pichia / Q000235:genetics; D021122:Protein Subunits; D011994:Recombinant Proteins / Q000032:analysis / Q000235:genetics; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D014357:Trypsin / Q000737:chemistry / Q000378:metabolism
chemical_list: D001427:Bacterial Toxins; D004220:Disulfides; D004768:Enterotoxins; D029968:Escherichia coli Proteins; D021122:Protein Subunits; D011994:Recombinant Proteins; C027538:heat-labile enterotoxin, E coli; D014357:Trypsin