Comparison of IMAC and MOAC for phosphopeptide enrichment by column chromatography

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.

► IMAC-Fe3+- and TiO2-column chromatography were compared.
► Specificity of IMAC-Fe3+ increases when 0.1 M TFA is used instead of 0.1 M acetic acid.
► Fe3+ is released for loading/washing step when 0.1 M TFA is used as loading buffer.
► Fe3+ precipitates for elution of phosphopeptides with NH4OH.
► IMAC-Fe3+ is not a convenient media for repetitive use on the contrary to TiO2.