Evaluation of the impact of some experimental procedures on different phosphopeptide enrichment techniques.

The complete characterization of phosphorylated proteins requires an efficient procedure for the enrichment of phosphopeptides from amongst a complicated peptide mixture. The sensitivity of the traditional immobilized metal affinity chromatography (IMAC) approach is severely affected by various buffers, detergents and other reagents normally utilized in biochemical and cell biological procedures, and thus pre-purification steps such as reversed-phase chromatography is required prior to phosphopeptide enrichment. Here we evaluate the use of different 'non-phosphopeptide-excluding compounds' in the loading buffer for titanium dioxide (TiO(2)) chromatography and show that TiO(2) is more robust and tolerant towards many reagents, including salts, detergents and other low molecular mass molecules, than conventional IMAC. In addition, we show that the inclusion of various detergents can enhance the efficiency of this enrichment method, as phosphopeptides that otherwise adhere to plastic surfaces can be efficiently solubilized and subsequently purified. The TiO(2) chromatography technique is also compared to zirconium dioxide chromatography for phosphopeptide enrichment. Copyright 2007 John Wiley & Sons, Ltd.