文摘
Glycosphingolipids (GSLs), composed of a hydrophiliccarbohydrate chain and a lipophilic ceramide anchor, playpivotal roles in countless biological processes, includingthe development of cancer. As part of the investigation ofthe vertebrate glycome, GSL analysis is undergoing rapidexpansion owing to the application of modern massspectrometry. Here we introduce direct coupling of IR-MALDI-o-TOF mass spectrometry with the TLC overlaybinding 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 ofprotein-detected GSLs. The high specificity and sensitivityis demonstrated by use of antibodies, bacterial toxins, anda plant lectin. The procedure works on a nanogram scale,and detection limits of less than 1 ng at its best ofimmunostained GSLs were obtained. Furthermore, onlycrude lipid extracts of biological sources are required forTLC-IR-MALDI-MS, omitting any laborious GSL downstream purification procedures. This strategy was successfully applied to the identification of cancer-associatedGSLs in human hepatocellular and pancreatic tumors.Thus, the in situ TLC-IR-MALDI-MS of immunolabeledGSLs opens new doors by delivering specific structuralinformation of trace quantities of GSLs with only a limitedinvestment in sample preparation.