title: Comparison of IMAC and MOAC for phosphopeptide enrichment by column chromatography.
abstract: Automated phosphopeptide enrichment prior to MS analysis by means of Immobilized Metal Affinity Chromatography (IMAC) and Metal Oxide Affinity Chromatography (MOAC) has been probed with packed columns. We compared POROS-Fe³⁺ and TiO₂ (respectively IMAC and MOAC media), using a simple mixture of peptides from casein-albumin and a complex mixture of peptides isolated from mouse liver. With theses samples, selectivity of POROS-Fe³⁺ and TiO₂ were pH dependant. In the case of liver extract, selectivity increased from 12-18% to 58-60% when loading buffer contained 0.1 M acetic acid or 0.1 M trifluoroacetic acid, respectively. However, with POROS-Fe³⁺ column, the number of identifications decreased from 356 phosphopeptides with 0.1 M acetic acid to 119 phosphopeptides with 0.1 M TFA. This decrease of binding capacity of POROS-Fe³⁺ was associated with strong Fe³⁺ leaching. Furthermore, repetitive use of IMAC-Fe³⁺ with the 0.5 M NH₄OH solution required for phosphopeptide elution induced Fe₂O₃ accumulation in the column. By comparison, MOAC columns packed with TiO₂ support do not present any problem of stability in the same conditions and provide a reliable solution for packed column phosphopeptide enrichment.
authors.affiliation: UMR 5248, CNRS-UBx1-IPB, Centre de Génomique Fonctionnelle, Université de Bordeaux, BP 68, 146 Rue Léo Saignat, 33076 Bordeaux, France. l.negroni@cbmn.u-bordeaux.fr
authors.forename: Luc
authors.initials: L
authors.lastname: Negroni
authors.forename: Stephane
authors.initials: S
authors.lastname: Claverol
authors.forename: Jean
authors.initials: J
authors.lastname: Rosenbaum
authors.forename: Eric
authors.initials: E
authors.lastname: Chevet
authors.forename: Marc
authors.initials: M
authors.lastname: Bonneu
authors.forename: Jean-Marie
authors.initials: JM
authors.lastname: Schmitter
mesh_terms: D002846:Chromatography, Affinity / Q000379:methods*; D010748:Phosphopeptides / Q000302:isolation & purification*; D053719:Tandem Mass Spectrometry
chemical_list: D010748:Phosphopeptides