Direct analysis of silica gel extracts from immunostained glycosphingolipids by nanoelectrospray ionization quadrupole time-of-flight mass spectrometry.
A combined strategy of preparative high-performance thin-layer chromatography overlay assay and mass spectrometry was established for the structural characterization of immunostained glycosphingolipids (GSLs) in silica gel extracts. Crude chloroform/methanol/water (30/60/8, v/v/v) extracts of immunostained TLC bands were analyzed by nanoelectrospray low-energy CID mass spectrometry without further purification. The GSL species investigated were isomeric monosialogangliosides of the neolacto series from a ganglioside preparation of human granulocytes, the disialoganglioside GD3 from a human melanoma lipid extract, and ganglio series Gg3Cer of a neutral GSL preparation from murine lymphoreticular MDAY-D2 cells. For the specific detection of lipid-bound oligosaccharides, polyclonal chicken IgY, murine monoclonal IgG3, and IgM antibodies were used. The resulting mass spectra show that only analytical quantities of approximately 1 microg of a single GSL within a complex mixture are required for the structure determination of immunostained GSLs by MS and MS/MS. All species investigated were detected as singly charged deprotonated molecular ions, and neither buffer-derived salt adducts nor coextracted contaminants from the immunostaining procedure or the silica gel layer were observed. This effective HPTLC-MS-joined procedure offers a wide range of applications for any carbohydrate binding agents such as bacterial toxins, plant lectins, and others.
Meisen
Iris
I
Institute for Medical Physics and Biophysics, University of Münster, Robert-Koch-Strasse 31, D-48149 Münster, Germany.
Peter-Katalinić
Jasna
J
Müthing
Johannes
J
D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry; D002855:Chromatography, Thin Layer / Q000379:methods; D005732:Gangliosides / Q000032:analysis; D006028:Glycosphingolipids / Q000032:analysis* / Q000737:chemistry / Q000302:isolation & purification*; D006098:Granulocytes / Q000737:chemistry; D006801:Humans; D007150:Immunohistochemistry / Q000379:methods; D008055:Lipids / Q000737:chemistry; D008214:Lymphocytes / Q000737:chemistry; D008545:Melanoma / Q000737:chemistry* / Q000276:immunology; D015394:Molecular Structure; D009844:Oligosaccharides / Q000032:analysis / Q000737:chemistry; D058428:Silica Gel; D012822:Silicon Dioxide / Q000737:chemistry*; D021241:Spectrometry, Mass, Electrospray Ionization / Q000295:instrumentation / Q000379:methods*; D013844:Thiazoles / Q000737:chemistry
D000911:Antibodies, Monoclonal; D005732:Gangliosides; D006028:Glycosphingolipids; D008055:Lipids; D009844:Oligosaccharides; D013844:Thiazoles; D058428:Silica Gel; D012822:Silicon Dioxide; C035646:primuline