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Glycobiology Advance Access published online on September 15, 2004
Glycobiology, doi:10.1093/glycob/cwh149
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Received September 25, 2003
Revised September 9, 2004
Accepted September 10, 2004

ORIGINAL ARTICLES

A robust and selective method for the quantification of glycosylphosphatidylinositols in biological samples

James I. MacRae 1 and Michael A. J. Ferguson 1*

1 The Division of Biological Chemistry & Molecular Microbiology, The School of Life Sciences, The Wellcome Trust Biocentre, The University of Dundee, Dundee DD1 5EH, Scotland, UK

* To whom correspondence should be addressed. E-mail: m.a.j.ferguson{at}dundee.ac.uk.


  Abstract

We have developed an assay for the quantification of glycosylphosphatidylinositol (GPI)-anchored glycoconjugates. The method is based on nitrous acid deamination and sodium borodeuteride reduction of the glucosamine residue, common to all GPI structures, to yield [1-2H]-2,5-anhydromannitol. Detection, following acid methanolysis and trimethylsilyl derivatisation, is by selected ion monitoring gas chromatography-mass spectrometry. The unnatural inositol isomer scyllo-inositol is used as an internal standard and the [1-2H]-2,5-anhydromannitol trimethylsilyl derivative is detected by following a characteristic electron-impact fragment ion at m/z 273. This method is more selective for GPIs than assays based on measuring myo-inositol content, which are often confounded by contaminating inositol-phospholipids. We show that the method can be applied to measure total GPI content in crude total lipid extracts and even in whole trypanosome ghosts. The method was applied to whole cell lysates of wild type, GPI-deficient and glycosaminoglycan-deficient CHO cells. The data revealed that proteoglycans did not interfere with total glucosamine estimates but that there are background levels of non-GPI and non-proteoglycan glucosamine-containing material in CHO cells.

Keywords: glycosylphosphatidylinositol; GPI; trypanosome; glucosamine; inositol.
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