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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
Author(s) -
Müsken Anne,
Souady Jamal,
Dreisewerd Klaus,
Zhang Wenlan,
Distler Ute,
PeterKatalinić Jasna,
MillerPodraza Halina,
Karch Helge,
Müthing Johannes
Publication year - 2010
Publication title -
rapid communications in mass spectrometry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.528
H-Index - 136
eISSN - 1097-0231
pISSN - 0951-4198
DOI - 10.1002/rcm.4480
Subject(s) - chemistry , chromatography , mass spectrometry , bacteria , matrix assisted laser desorption/ionization , desorption , adsorption , organic chemistry , genetics , biology
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. Copyright © 2010 John Wiley & Sons, Ltd.