LC-MS analysis of pig intestine sulfatides: Interaction withEscherichia coliSTb enterotoxin and characterization of molecular species present

Abstract

STb, a 48-amino acid thermostable enterotoxin is produced by enterotoxigenic Escherichia coli strains and is responsible for diarrheal diseases in many animals, including man. Our laboratory recently identified a family of molecules, from a lipid extract of porcine intestinal epithelial cells, that could bind to STb. These molecules were identified as sulfatides as they reacted with a monoclonal antibody raised against this family of molecules. However, as the epitope recognized by this monoclonal antibody was the galactose 3-sulfate, a doubt could remain as to the exact nature of the identified receptors. The goal of this study was thus to confirm the chemical nature of the STb-binding molecule as sulfatides or as distinctive molecules comprising a sulfated galactosyl residue. Using a thin-layer chromatography-overlay method we confirmed using antibodies to STb that STb recognizes the commercial sulfatides and a band migrating at the same level from the intestinal tissue lipid extract obtained from an 8-week-old piglet. The compounds recovered from the silica gel plates were analyzed by mass spectrometry in electrospray negative-ionization mode. The most abundant ions observed had m/z values of 779, 795, 879 and 907. For commercial bovine brain sulfatides the ions 795, 879 and 907 have been attributed to hydroxylated sulfatides with a saturated fatty acid chain containing 16, 22 and 24 carbons, while the 779 ion contained a saturated fatty acid chain of 16 carbons. The general profile of the ions observed was similar to the already described commercial bovine brain sulfatides.

Polymorphonuclear Leukocyte Apoptosis Is Accelerated by Sulfatides or Sulfatides-TreatedSalmonellaTyphimurium Bacteria

“…Sulfatides mediate the attachment of Pseudomonas aeruginosa to human epithelial cells [ 10 ]. Immunohistochemistry study detected sulfatides on the surface of mucosal epithelial cells, involved in the binding of Escherichia coli enterotoxin b [ 11 , 12 ].…”

Regioselective Cleavage of Myoglobin with Copper(II) Compounds at Neutral pH

“…Electrospray mass spectrometry (ESIMS) has been widely employed because of its ability to measure the exact molecular mass. The convenience of interfacing the ion source to the high performance separation techniques such as liquid chromatography (LC) has made great progress in its biological application. − In ESIMS, protonated molecules may be further analyzed by fragmentation using collision-induced dissociation (MS/MS) to obtain structural information. − The spectra of fragment ions produced by collision can be acquired to characterize the peptides with the known sequence. The reported MS/MS spectra, which gave out enough consecutive fragment ions, were limited to peptides with less than 20 amino acid residues. − So far, for bigger or medium sized peptides, MS/MS analysis mainly focuses on abundant multicharged fragments. − …”

“…The convenience of interfacing the ion source to the high performance separation techniques such as liquid chromatography (LC) has made great progress in its biological application. [34][35][36][37] In ESIMS, protonated molecules may be further analyzed by fragmentation using collision-induced dissociation (MS/MS) to obtain structural information. [38][39][40] The spectra of fragment ions produced by collision can be acquired to characterize the peptides with the known sequence.…”