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 Anne Mu ¨ sken 1 , Jamal Souady 2 , Klaus Dreisewerd 2 , Wenlan Zhang 1 , Ute Distler 1 , Jasna Peter-Katalinic ´ 2 , Halina Miller-Podraza 3 , Helge Karch 1 and Johannes Mu ¨ thing 1,4 * 1 Institute for Hygiene, University of Mu ¨nster, D-48149 Mu ¨nster, Germany 2 Institute of Medical Physics and Biophysics, University of Mu ¨nster, D-48149 Mu ¨nster, Germany 3 Institute of Medical Biochemistry, Go ¨teborg University, P.O. Box 440, SE 405 30 Go ¨teborg, Sweden 4 Interdisciplinary Center for Clinical Research (IZKF), University of Mu ¨nster, D-48149 Mu ¨nster, Germany Received 3 November 2009; Revised 6 January 2010; Accepted 23 January 2010 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 second- ary 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. Copyright # 2010 John Wiley & Sons, Ltd. Urinary tract infections are considered to be the most common bacterial infections in humans. 1,2 They develop from peri-urethral colonization, followed by ascension of the bacteria to the urethra and further to the bladder and kidney causing cystitis and acute pyelonephritis, respectively. Uropathogenic Escherichia coli often have P fimbriae which carry the receptor binding pyelonephritis-associated pili (Pap) G protein at its distal end. 3–5 PapG of class II mediates pathogen adherence in renal colonization and acute pyelonephritis 6 and binds to the neutral glycosphingolipids (GSLs) with Gala4Gal as an internal or terminal part including globotetraosylceramide (Gb4Cer or globoside, GalNAcb3Gala4Galb4Glcb1Cer) and globotriaosylceramide (Gb3Cer or CD77, Gala4Galb4Glcb1Cer), 7 both being abundant in the upper urinary tract of humans. 8 GSLs are composed of a hydrophilic oligosaccharide chain and a hydrophobic lipid anchor named ceramide. 9 In mammalian cells, the ceramide moiety is typically built up from the long-chain amino alcohol sphingosine (d18:1), which is linked with a fatty acid varying in the chain length from C16 to C24. Various types of pathogens exploit GSLs on host cells as membrane receptors, which contribute to colonization and virulence. 10 High-performance thin-layer chromatography (TLC) is the most common technique used for the separation of GSLs in mixtures. 11 GSL species can be differentiated in TLC overlay assays directly on the silica gel of the plate using carbohydrate-binding proteins such as antibodies and bacterial toxins. A considerable improvement was the coupling of the TLC overlay assay with infrared matrix- assisted laser desorption/ionization orthogonal time-of- flight mass spectrometry (IR-MALDI-o-TOF-MS) for the structural characterization of GSLs. 12–14 As a novelty of this strategy, we here describe the coupling of bacterial adhesion RAPID COMMUNICATIONS IN MASS SPECTROMETRY Rapid Commun. Mass Spectrom. 2010; 24: 1032–1038 Published online in Wiley InterScience (www.interscience.wiley.com) DOI: 10.1002/rcm.4480 *Correspondence to: J. Mu ¨ thing, Institute for Hygiene and Inter- disciplinary Center for Clinical Research (IZKF), University of Mu ¨ nster, D-48149 Mu ¨ nster, Germany. E-mail: jm@uni-muenster.de Copyright # 2010 John Wiley & Sons, Ltd.