Development of a titanium dioxide nanoparticle pipette-tip for the selective enrichment of phosphorylated peptides

The selective enrichment of specific proteins or peptides on micropipette tips prior to mass spectrometry analysis, which can minimize nonspecific interferences as well as sample loss, has been an important issue in current proteomics field. In this paper, we have developed an easy-to-use phosphopeptide- selective pipette tip in which titanium dioxide nanoparticles were embedded in monolithic structure photopolymerized from ethylene glycol dimethacrylate. The simple and convenient fabrication was feasible in a commercial polypropylene pipette tip. Phosphorylated peptides were isolated from non-phosphopeptides by TiO2 nanoparticle and eluted by 100 mM ammonium phosphate (pH 8.5), which was compatible with 2,5-dihydroxybenzoic acid (DHB)/1% phosphoric acid matrix and allowed for direct analysis of the elution fraction by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) without the necessity of desalting pretreatment. Tryptic digested a-casein and beta-casein spiked into bovine serum albumin (BSA) nonphosphorylated peptides (molar ratio 1: 1: 10) were used to assess the selectivity of TiO2 tips. The effect of 50 mM ammonium hydrogencarbonate, pH 8 in 50% acetonitrile used as a wash buffer in reduction of nonspecific bound peptide to TiO2 tip was dramatic. Almost all non-phosphopeptides were not detected by MALDI-MS analysis. The lowest detectable amount of phosphopeptide was estimated at low femtomole level. The easy-to-use TiO2-embeded tips operated in combination with the modified wash and elution conditions enable an efficient phosphopeptide enrichment for mass spectrometric analysis. (c) 2007 Elsevier B.V All rights reserved.