title: Matching IR-MALDI-o-TOF mass spectrometry with the TLC overlay binding assay and its clinical application for tracing tumor-associated glycosphingolipids in hepatocellular and pancreatic cancer.
abstract: Glycosphingolipids (GSLs), composed of a hydrophilic carbohydrate chain and a lipophilic ceramide anchor, play pivotal roles in countless biological processes, including the development of cancer. As part of the investigation of the vertebrate glycome, GSL analysis is undergoing rapid expansion owing to the application of modern mass spectrometry. Here we introduce direct coupling of IR-MALDI-o-TOF mass spectrometry with the TLC overlay binding assay for the structural characterization of GSLs. We matched three complementary methods including (i) TLC separation of GSLs, (ii) their detection with oligosaccharide-specific proteins, and (iii) in situ MS analysis of protein-detected GSLs. The high specificity and sensitivity is demonstrated by use of antibodies, bacterial toxins, and a plant lectin. The procedure works on a nanogram scale, and detection limits of less than 1 ng at its best of immunostained GSLs were obtained. Furthermore, only crude lipid extracts of biological sources are required for TLC-IR-MALDI-MS, omitting any laborious GSL downstream purification procedures. This strategy was successfully applied to the identification of cancer-associated GSLs in human hepatocellular and pancreatic tumors. Thus, the in situ TLC-IR-MALDI-MS of immunolabeled GSLs opens new doors by delivering specific structural information of trace quantities of GSLs with only a limited investment in sample preparation.
authors.affiliation: Institute of Medical Physics and Biophysics and Institute for Hygiene, University of Münster, D-48149 Münster, Germany.
authors.forename: Ute
authors.initials: U
authors.lastname: Distler
authors.forename: Marcel
authors.initials: M
authors.lastname: Hülsewig
authors.forename: Jamal
authors.initials: J
authors.lastname: Souady
authors.forename: Klaus
authors.initials: K
authors.lastname: Dreisewerd
authors.forename: Jörg
authors.initials: J
authors.lastname: Haier
authors.forename: Norbert
authors.initials: N
authors.lastname: Senninger
authors.forename: Alexander W
authors.initials: AW
authors.lastname: Friedrich
authors.forename: Helge
authors.initials: H
authors.lastname: Karch
authors.forename: Franz
authors.initials: F
authors.lastname: Hillenkamp
authors.forename: Stefan
authors.initials: S
authors.lastname: Berkenkamp
authors.forename: Jasna
authors.initials: J
authors.lastname: Peter-Katalinić
authors.forename: Johannes
authors.initials: J
authors.lastname: Müthing
mesh_terms: D002240:Carbohydrate Sequence; D002855:Chromatography, Thin Layer / Q000379:methods*; D006028:Glycosphingolipids / Q000378:metabolism*; D008113:Liver Neoplasms / Q000378:metabolism*; D008969:Molecular Sequence Data; D010190:Pancreatic Neoplasms / Q000378:metabolism*; D012680:Sensitivity and Specificity; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D013055:Spectrophotometry, Infrared / Q000379:methods*
chemical_list: D006028:Glycosphingolipids