Proteomic scale high-sensitivity analyses of GPI membrane anchors.
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.
Mehlert
Angela
A
Division of Biological Chemistry and Drug Discovery, College of Life Sciences, Wellcome Trust Biocentre, University of Dundee, Dundee DD1 5EH, UK.
Ferguson
Michael A J
MA
D000818:Animals; D008401:Gas Chromatography-Mass Spectrometry; D017261:Glycosylphosphatidylinositols / Q000032:analysis* / Q000737:chemistry; D007294:Inositol / Q000032:analysis; D013058:Mass Spectrometry; D040901:Proteomics / Q000379:methods*; D014346:Trypanosoma brucei brucei / Q000378:metabolism
D017261:Glycosylphosphatidylinositols; D007294:Inositol