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 non-specific 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 α-casein and β-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.