title: Application of thin-layer chromatography/infrared matrix-assisted laser desorption/ionization orthogonal time-of-flight mass spectrometry to structural analysis of bacteria-binding glycosphingolipids selected by affinity detection.
abstract: Glycosphingolipids (GSLs) play key roles in the manifestation of infectious diseases as attachment sites for pathogens. The thin-layer chromatography (TLC) overlay assay represents one of the most powerful approaches for the detection of GSL receptors of microorganisms. Here we report on the direct structural characterization of microbial GSL receptors by employment of the TLC overlay assay combined with infrared matrix-assisted laser desorption/ionization orthogonal time-of-flight mass spectrometry (IR-MALDI-o-TOF-MS). The procedure includes TLC separation of GSL mixtures, overlay of the chromatogram with GSL-specific bacteria, detection of bound microbes with primary antibodies against bacterial surface proteins and appropriate alkaline phosphatase labeled secondary antibodies, and in situ MS analysis of bacteria-specific GSL receptors. The combined method works on microgram scale of GSL mixtures and is advantageous in that it omits laborious and time-consuming GSL extraction from the silica gel layer. This technique was successfully applied to the compositional analysis of globo-series neutral GSLs recognized by P-fimbriated Escherichia coli bacteria, which were used as model microorganisms for infection of the human urinary tract. Thus, direct TLC/IR-MALDI-o-TOF-MS adds a novel facet to this fast and sensitive method offering a wide range of applications for the investigation of carbohydrate-specific pathogens involved in human infectious diseases.
authors.affiliation: Institute for Hygiene, University of Münster, D-48149 Münster, Germany.
authors.forename: Anne
authors.initials: A
authors.lastname: Müsken
authors.forename: Jamal
authors.initials: J
authors.lastname: Souady
authors.forename: Klaus
authors.initials: K
authors.lastname: Dreisewerd
authors.forename: Wenlan
authors.initials: W
authors.lastname: Zhang
authors.forename: Ute
authors.initials: U
authors.lastname: Distler
authors.forename: Jasna
authors.initials: J
authors.lastname: Peter-Katalinić
authors.forename: Halina
authors.initials: H
authors.lastname: Miller-Podraza
authors.forename: Helge
authors.initials: H
authors.lastname: Karch
authors.forename: Johannes
authors.initials: J
authors.lastname: Müthing
mesh_terms: D000907:Antibodies, Bacterial / Q000378:metabolism; D001422:Bacterial Adhesion; D002855:Chromatography, Thin Layer / Q000379:methods*; D020650:Combinatorial Chemistry Techniques / Q000379:methods; D004912:Erythrocytes / Q000737:chemistry; D010861:Fimbriae, Bacterial / Q000378:metabolism; D005915:Globosides / Q000378:metabolism; D006028:Glycosphingolipids / Q000378:metabolism*; D006801:Humans; D007124:Immunoenzyme Techniques / Q000379:methods*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D014281:Trihexosylceramides / Q000378:metabolism; D014552:Urinary Tract Infections / Q000097:blood / Q000382:microbiology; D056689:Uropathogenic Escherichia coli / Q000378:metabolism*
chemical_list: D000907:Antibodies, Bacterial; D005915:Globosides; D006028:Glycosphingolipids; D014281:Trihexosylceramides; C024032:globotetraosylceramide; C018549:globotriaosylceramide