Detection of Glycolipids as Biotinylated Derivatives Using Enhanced Chemiluminescence

Hernández, Luís E.; Brewin, N. J.; Drøbak, Bjørn K.

doi:10.1006/abio.1996.0378

Cited by 5 publications

(3 citation statements)

References 7 publications

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“…However, in these cases the only available hydroxyl groups are part of a five-or six-membered ring and, therefore, are more difficult to oxidize. Thus, higher concentrations of periodate are required (16,17). At these higher concentrations permeation of the bilayer can be a problem (18).…”

mentioning

confidence: 99%

Assay for the transbilayer distribution of glycolipids: selective oxidation of glucosylceramide to glucuronylceramide by TEMPO nitroxyl radicals

Sillence

Raggers

Neville

et al. 2000

Journal of Lipid Research

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In the present study, 2,2,6,6-tetramethylpiperidinooxy nitroxide (TEMPO) has been applied successfully to discriminate between glucosylceramide in the outer and inner leaflets of closed membrane bilayers. The nitroxyl radicals TEMPO and carboxy-TEMPO, once oxidized to nitrosonium ions, are capable of oxidizing residues that contain primary hydroxyl and amino groups. When applied to radiolabeled glucosylceramide in liposomes, oxidation with TEMPO led to an oxidized product that was easily separated from the original lipid by thin-layer chromatography, and that was identified by mass spectrometric analysis as the corresponding acid glucuronylceramide. To test whether oxidation was confined to the external leaflet, TEMPO was applied to large unilamellar vesicles (LUVs) consisting of egg phosphatidylcholine-egg phosphatidylethanolamine-cholesterol 55:5:40 (mol/mol). TEMPO oxidized most radiolabeled phosphatidylethanolamine, whereas carboxy-TEMPO oxidized only half. Hydrolysis by phospholipase A 2 confirmed that 50% of the phosphatidylethanolamine was accessible in the external bilayer leaflet, suggesting that TEMPO penetrated the lipid bilayer and carboxy-TEMPO did not. When applied to LUVs containing Ͻ 1 mol% radiolabeled glucosylceramide or short-chain C 6 -glucosylceramide, carboxy-TEMPO oxidized half the glucosylceramide. However, if surface C 6 -glucosylceramide was first depleted by bovine serum albumin (BSA) (extracting 49 ؎ 1%), 94% of the remaining C 6 -glucosylceramide was resistant to oxidation. Carboxy-TEMPO oxidized glucosylceramide on the surface of LUVs without affecting inner leaflet glucosylceramide. At pH 9.5 and at 0 ؇ C, the reaction reached completion by 20 min.

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confidence: 99%

Assay for the transbilayer distribution of glycolipids: selective oxidation of glucosylceramide to glucuronylceramide by TEMPO nitroxyl radicals

Sillence

Raggers

Neville

et al. 2000

Journal of Lipid Research

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“…When the glycolipids on the PVDF membrane are mildly oxidized, they can be conjugated with streptavidin-horseradish peroxidase and, using enhanced chemiluminescence, are clearly visible at a concentration factor of 10 lower than detected by the orcinol reagent. 16 The method can be generally applied to the use of a specific antibody for detection and, since the staining procedure is nondestructive, the glycolipids can still be used for further molecular analyses.…”

Section: Thin-layer Chromatographic Methodsmentioning

confidence: 99%

Glycolipid Analysis

Morrison¹

2000

Encyclopedia of Analytical Chemistry

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Glycolipids are conjugated macromolecules that have a carbohydrate (oligosaccharide) component covalently bound to a lipid (diacylglycerol, ceramide, sterol or polyprenol) component. They are present both in procaryotes and eucaryotes but the carbohydrate components, as well as the lipid components, vary considerably between different animal, plant and microbial cells. The biological specificity of a glycolipid is due mainly to the carbohydrate structure. The amphipathic nature of glycolipids means that a single extraction scheme is impractical for all the different molecular species while special techniques are required to differentiate between the neutral glycolipids and acidic glycolipids (gangliosides). Thin‐layer chromatography (TLC) and high‐performance liquid chromatography (HPLC) are the major purification methods. Chemical methods are used to determine the carbohydrate and lipid profiles as well as the linkage analysis of both types of components and are used in conjunction with gas chromatography (GC), HPLC and TLC, sometimes coupled with mass spectroscopy (MS). Enzymatic methods can be used to determine the linkage sequence and anomeric configurations of the oligosaccharide structures and the same parameters are being determined by immunological methods with increasing frequency. Enzymatic methods are also used to establish the linkage analysis of the fatty acid in the diacylglycerol species. However, various MS and both 1 H‐ and 13 C‐NMR (nuclear magnetic resonance) methods, but mostly the former, are now invariably used to confirm the full structural features of an unknown glycolipid. The type of MS method used, matrix‐assisted laser desorption/ionization (MALDI), fast atom bombardment (FAB) or tandem mass spectrometry (MS/MS) will depend on the type of glycolipid.

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“…It is a negative-sense RNA virus and has three generas: influenza A virus, influenza B virus, influenza C virus. As a key viral envelop glycoprotein [98], HA can bind specifically to sialic acid-containing receptors on the host cell surface and enables virus to attach to the targeted host cells [99,100]. In the last century, there were some typical outbreaks of influenza A virus subtypes, such as the H1N1 Spanish Flu [94], the H2N2 Asian Flu [95], the H3N2 and H5N1 Hong Kong Flu [96].…”

Section: Influenza Virusmentioning

confidence: 99%

Mass spectrometry based proteomic studies on viruses and hosts – A review

Zheng

Sugrue

Tang

2011

Analytica Chimica Acta

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In terms of proteomic research in the 21st century, the realm of virology is still regarded as an enormous challenge mainly brought by three aspects, namely, studying on the complex proteome of the virus with unexpected variations, developing more accurate analytical techniques as well as understanding viral pathogenesis and virus-host interaction dynamics. Progresses in these areas will be helpful to vaccine design and antiviral drugs discovery. Mass spectrometry based proteomics have shown exceptional display of capabilities, not only precisely identifying viral and cellular proteins that are functionally, structurally, and dynamically changed upon virus infection, but also enabling us to detect important pathway proteins. In addition, many isolation and purification techniques and quantitative strategies in conjunction with MS can significantly improve the sensitivity of mass spectrometry for detecting low-abundant proteins, replenishing the stock of virus proteome and enlarging the protein-protein interaction maps. Nevertheless, only a small proportion of the infectious viruses in both of animal and plant have been studied using this approach. As more virus and host genomes are being sequenced, MS-based proteomics is becoming an indispensable tool for virology. In this paper, we provide a brief review of the current technologies and their applications in studying selected viruses and hosts.

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Detection of Glycolipids as Biotinylated Derivatives Using Enhanced Chemiluminescence

Cited by 5 publications

References 7 publications

Assay for the transbilayer distribution of glycolipids: selective oxidation of glucosylceramide to glucuronylceramide by TEMPO nitroxyl radicals

Assay for the transbilayer distribution of glycolipids: selective oxidation of glucosylceramide to glucuronylceramide by TEMPO nitroxyl radicals

Glycolipid Analysis

Mass spectrometry based proteomic studies on viruses and hosts – A review

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