High-pH anion-exchange chromatographic analysis of phosphorylated compounds: application to isolation and characterization of nonpeptide mycobacterial antigens

Analytical Biochemistry
Y PoquetJ J Fournié

Abstract

A rapid and sensitive high-pH anion-exchange chromatography (HPAEC) method for the separation and quantification of phosphorylated antigens in mycobacterial extracts has been developed. This method provides the separation of mono-, di-, or triphosphonucleotides and of various other phosphorylated molecules. Dual detection by conductimetry and UV absorption downstream of a chemical suppressor constitute nondegradative and highly sensitive tools for the physical detection and the quantification of phosphorylated compounds in biological samples. The lower limit of accurate quantification is around 1 nmol per sample. This method was used for the separation of several phosphorylated antigens activating human gamma delta T lymphocytes from semipurified mycobacterial fractions. Their quantification revealed that the minimal concentration activating a gamma delta T cell clone is between 1 and 5 nM. This approach can be used for more general preparative purposes with samples where minute amounts of biologically active phosphoanions are analyzed.

Citations

Feb 8, 2011·Archivum Immunologiae Et Therapiae Experimentalis·Eric Champagne
Sep 7, 2000·FASEB Journal : Official Publication of the Federation of American Societies for Experimental Biology·C BelmantJ J Fournié
Oct 25, 2001·The Journal of Biological Chemistry·Juliane FeurleMartin Wilhelm
Nov 5, 1999·The Journal of Biological Chemistry·C BelmantJ J Fournié
Sep 25, 2004·Immunology Letters·Mary Poupot, Jean-Jacques Fournié

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