Selective TiO₂ Phosphopeptide Enrichment of Complex Samples in the Nanogram Range

Abstract

Phosphopeptide enrichment is a commonly used sample preparation step for investigating phosphorylation. TiO2-based enrichment has been demonstrated to have excellent performance both for large amounts of complex and for small amounts of simple samples. However, it has not yet been studied for complex samples in the nanogram range. Our objective was to develop a methodology applicable for complex samples in the low nanogram range, useful for mass spectrometry analysis of tissue microarrays. The selectivity and performance of two stationary phases (TiO2 nanoparticle-coated monolithic column and spin tip filled with TiO2 microspheres) and several loading solvents were studied. Based on this study, we developed an effective and robust method, based on a spin tip with a non-conventional 50 mM citric acid-based loading solvent. It gave excellent results for phosphopeptide enrichment from samples containing a few nanograms of a complex protein mixture.

Keywords

• solid-phase extraction
• phosphopeptide enrichment
• phosphopeptide isolation
• phosphorylation
• titanium dioxide
• metal oxide affinity chromatography