title: High-pH anion-exchange chromatographic analysis of phosphorylated compounds: application to isolation and characterization of nonpeptide mycobacterial antigens.
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.
authors.affiliation: INSERM U395, CHU Purpan, Toulouse, France.
authors.forename: Y
authors.initials: Y
authors.lastname: Poquet
authors.forename: P
authors.initials: P
authors.lastname: Constant
authors.forename: M A
authors.initials: MA
authors.lastname: Peyrat
authors.forename: R
authors.initials: R
authors.lastname: Poupot
authors.forename: F
authors.initials: F
authors.lastname: Halary
authors.forename: M
authors.initials: M
authors.lastname: Bonneville
authors.forename: J J
authors.initials: JJ
authors.lastname: Fournié
mesh_terms: D000942:Antigens, Bacterial / Q000302:isolation & purification*; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange / Q000379:methods*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D009161:Mycobacterium; D010766:Phosphorylation
chemical_list: D000942:Antigens, Bacterial