Sequential Fe3O4/TiO2 enrichment for phosphopeptide analysis by liquid chromatography/tandem mass spectrometry

Abstract

Protein phosphorylation regulates a wide range of cellular functions and is associated with signaling pathways in cells. Various strategies for enrichment of phosphoproteins or phosphopeptides have been developed. Here, we developed a novel sequential phosphopeptide enrichment method, using magnetic iron oxide (Fe3O4) and titanium dioxide (TiO2) particles, to detect mono- and multi-phosphorylated peptides. In the first step, phosphopeptides were captured on Fe3O4 particles. In a subsequent step, any residual phosphopeptides were captured on TiO2 particles. The particles were eluted and rinsed to yield phosphopeptide-enriched fractions that were combined and analyzed using liquid chromatography/tandem mass spectrometry (LC/MS/MS). The validity of this sequential Fe3O4/TiO2 enrichment strategy was demonstrated by the successful enrichment of bovine casein phosphopeptides. We then applied the sequential Fe3O4/TiO2 enrichment method to the analysis of phosphopeptides in L6 muscle cell lysates and successfully identified mono- and multi-phosphorylated peptides. Copyright (C) 2010 John Wiley & Sons, Ltd.