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Simultaneous profiling of individual glycosphingolipids in crude lipid extracts Despite the difficulties of studying glycosphingolipids (GSLs), they are of particular interest because changes in cellsurface glycosylation can signal important cellular changes, such as a transformation to malignancy. Johannes Mu¨thing, Jamal Souady, and co-workers at the University of Mu¨nster (Germany) report in a recent AC article (2009, DOI 10.1021/ac901948h) the refinement of a combination of three techniquesOTLC, immunodetection and staining, and MALDI MSOfor studying GSLs. The approach optimizes the use of the limited amount of sample material, and the researchers demonstrate its immediate applicability by contrasting healthy and cancerous liver tissues. Dapeng Zhou of the University of Texas M.D. Anderson Cancer Center recognizes the value of the refined analytical technique. “Glycosphingolipids as a class are one of the least understood biomacromolecules, due to a lack of appropriate technologies,” he says. “Coupling TLC immunostaining with [MS] is not new, as the authors note, but they take it a step further, showing that it works in measuring glycosphingolipids from surgical specimens.” GSLs are a subcategory of glycolipids and include three linked subunits: (i) a saccharide of varying structure, (ii) the amino alcohol sphingosine (itself a lipid), and (iii) a fatty acid of varying structure. With this combination of hydrophilic and hydrophobic pieces, GSLs are frequently found decorating a cell’s surface; the lipid serves as an anchor within the cell membrane, and the hydrophilic glycan is displayed in the extracellular space. In the Mu¨nster team’s process, a mixture of GSLs in a lipid extract is first separated by TLC to spatially resolve individual GSLs. Then, using “specific antiGSL antibodies, followed by visualization of enzyme-catalyzed staining at the sites of bound antibodiesOan adaptation of ELISAOindividual GSLs within the mix9536
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ture are detected,” says Souady. “The successive use of various antibodies with certain oligosaccharide specificities in one and the same chromatographic run represents the basic novelty of the procedure.” The group carefully selected enzyme-linked substrates to generate three distinctly colored, water-insoluble chromophores that are suitable for densitometric quantitation.
An experiment demonstrating the distinction between normal (N) and cancerous (T) liver cells. The TLC plate separated GSLs, which were then visualized by sequential ELISA-type stainings with three different targets and color outputs. Each band was then structurally characterized further via direct IR-MALDI MS.
The immunostaining thus offers information about the saccharides and their glycosidic linkagesOand without inhibiting further MS analysis. “The mass spectrometric investigation of intact GSLs is feasible because the immunostaining approach is nondestructive detectionOunlike conventional chemical staining of sugars or lipids,” says Souady. The ensuing MALDI MS reveals molecular mass information about the entire GSL, which can be used to deduce the nature of the fatty acid moiety. “The previously reported method leaves the majority of GSLs unexamined,” says Souady. “With multiple immunostaining of the same chromatogram, additional information on the composition of an unknown GSL
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mixture is obtained. This is of particular importance if only small samplesOfor instance, biopsiesOare available, thereby limiting the number of possible assays.” Akemi Suzuki of Tokai University (Japan) lauds the frugal nature of the technique, saying that it should be “very useful when we have a limited amount of sample.” He notes, however, that “the procedure will need to be optimized each time when using different antibodies or different samples containing glycolipids of very different concentration. Then, I might try to use several lanes of TLC, one for each antibody.” “This technology relies on existing antibodies specific for individual glycosphingolipids, so it will not identify unknown glycosphingolipid structures because the antibodies are not available,” adds Zhou. “The authors have mentioned that they are coupling the infrared MALDI source to other mass spectrometers with fragmentation capacities. That should allow them to identify candidates of new glycosphingolipid structures and to discriminate regioisomers.” Many future refinements are envisioned. “The investigation of the glycosphingolipidome is part of the glycome approach, aimed at unraveling the glycocode of certain mammalian cells or cell types,” says Souady. “Thus, the investigation of the glycosphingolipidome requires the analysis of a sample in its totality, as it is done when we work with total lipid extracts. Multiple TLC immunostaining combined with MS represents a first step forward, but we have to further improve even this novel technology, which is still in its infancy.” —Steven C. Powell
10.1021/AC902435A 2009 AMERICAN CHEMICAL SOCIETY
Published on Web 11/11/2009
