Proteomic scale high-sensitivity analyses of GPI membrane anchors |
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Authors: | Angela Mehlert Michael A J Ferguson |
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Institution: | (1) Division of Biological Chemistry and Drug Discovery, College of Life Sciences, Wellcome Trust Biocentre, University of Dundee, Dundee, DD1 5EH, UK; |
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Abstract: | Glycosylphosphatidylinositol (GPI) anchored proteins are ubiquitous in eukaryotic cells. Earlier analysis methods required
large amounts of purified protein to elucidate the structure of the GPI. This paper describes methods for analyzing GPIs on
a ‘proteomic’ scale. Partially purified proteins may be run on sodium dodecyl sulphate polyacrylamide gel electrophoresis
and then blotted onto a polyvinylidene difluoride (PVDF) membrane. Following identification of the protein the piece of PVDF
may be subjected to various chemical treatments, which are specific for GPI structures. The first method uses gas chromatography–mass
spectrometry and it enables the presence of a GPI anchor to be confirmed. The second method depends on the cleavage of phosphate
bonds and permits the carbohydrate structure to be elucidated by electrospray or matrix assisted laser desorption ionization-time
of flight mass spectrometry. The final method described uses deamination of the glucosamine residue to release the lipid moiety
for analysis by mass spectrometry. |
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Keywords: | |
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