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Identifying glycosphingolipids in tumors
Intensity (a.u.)
By combining three simple and responding disodiated molecular [M + 2Na – H] + well-known techniques, Johannes ions,” Müthing says. IV 6 nLc4Cer (CD75s) 1672.92/1674.93 α6 β4 β3 β4 β1 Müthing and colleagues at the Next, the researchers analyzed Cer University of Münster (Germany) mixtures of gangliosides, which have developed a sensitive and are negatively charged because specific method for detecting and they contain one or more sialic 1670 m/z 1680 structurally characterizing glyacid moieties. “All monosialylated Separation N T Laser cosphingolipids (GSLs) in small CD75s- and iso-CD75s-gangliobiological samples. GSLs, which sides preferentially appeared as consist of a hydrophilic sugar doubly sodiated molecular ions, chain on a hydrophobic ceramide, accompanied by minor monoDetection play important roles in biology sodiated molecular ions and less IR-MALDI and disease. “A major concern was intensive ions of desialylated ganto develop a strategy avoiding laDirect TLC-IR-MALDI MS of immunodetected CD75sgliosides,” Müthing says. Moreborious isolation and purification ganglioside (~2 ng) in a human liver tumor (T), showing over, even those isomers could be steps,” Müthing says. The work enhanced expression compared with normal tissue (N). differentiated from each other by is described in AC (2008, 80, The disodiated molecular ions at m/z 1672.92/1674.93 combining the TLC overlay assay 1835–1846). represent the tumor-associated CD75s-ganglioside with with IR-MALDI MS. SurprisThe three techniques are TLC, the structure Neu5Acα6Galβ4GlcNAcβ3Galβ4Glcβ1Cer ingly, gangliosides with only C16 an ELISA on the chromatography (d18:1, C24:1/C24:0). fatty acids in their ceramide moiplates, and MALDI MS. To those ety produced more intense signals standard methods, the researchers added orcinol, a phenolic compound from than those with C24 fatty acids. in situ ionization with an IR laser and lichens. Companion plates were overThe clinical studies were performed MS of immunostained GSLs. laid with a substance specific for GSLs, on crude liver and pancreatic tumor exStarting with reference spectra of such as an antibody, toxin, or lectin. tracts treated with sodium hydroxide to well-characterized GSLs, the researchers After incubation and washing, the saponify lipids other than GSLs. Comdetected as little as ~0.6 ng of neutral companion plates were overlaid with a pared with normal tissue, one patient’s GSLs and ~1.2 ng of gangliosides (negsecondary detection agent, such as an liver tumor expressed enhanced levels of atively charged GSLs). Moreover, they anti-immunoglobulin antibody, antiseveral CD75s-gangliosides. Iso-CD75sanalyzed crude lipid extracts from surtoxin, or antilectin antibody. “These gangliosides were overexpressed by a gical tumor specimens ranging from 45 different approaches were used to show second patient’s liver tumor and another to 264 mg wet weight. “These promisthat our procedure works with all the patient’s pancreatic tumor. A fourth ing results open the possibility to invescarbohydrate binding proteins so far patient’s pancreatic tumor displayed very tigate biopsy samples aimed at the iden- investigated, independently of biologihigh levels of the Shiga toxin receptor, a tification of tumor-associated GSLs at cal origin and the molecular size of the neutral GSL. “At some point, it might the early stage of cancer development, proteins used,” Müthing says. be possible to selectively destroy tumor because most human cancer cells are abUnfortunately, the IR laser vaporized cells using monoclonal antibodies, bacnormally glycosylated,” Müthing says. the plastic used to stabilize the silica gel, terial toxins, or toxic lectins,” Müthing The researchers prepared the refercrowding the spectra with polymeric says. “Therefore, identifying tumor-asence mixtures of neutral GSLs from ions. So, the researchers now wash off sociated GSLs could help oncologists human erythrocytes, granulocytes, or the plastic with chloroform. They chose tailor treatments to individual patients.” a mouse cell line and purchased human an IR laser, rather than a UV laser, beIn this case, the structures of the brain gangliosides. The mixtures were cause the IR laser also ionizes material ceramide moieties of the GSLs were applied to TLC plates coated with silica from the deeper layers of silica gel. not established unequivocally. “But our gel and separated in solvents composed The spectra produced by neutral study shows that it is possible to detect of chloroform, methanol, and water. GSLs showed that mass accuracy was and characterize GSLs associated with The plates were then treated with plashigh regardless of the type of antibody tumors,” Müthing says, “and that our tic to prevent the silica gel from flaking used. “All neutral GSL species primarprotocol can be used on milligrams of off during immunodetection. ily appeared as monosodiated molecutissue containing only nanograms of To detect the GSL spots, the relar ions in the positive-ion-mode mass GSLs.” a searchers stained one set of plates with spectra, accompanied by minor cor—Linda Sage © 2008 AMERICAN CHEMICAL SOCIET Y
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