Analysis of protein phosphorylation by monolithic extraction columns based on poly(divinylbenzene) containing embedded titanium dioxide and zirconium dioxide nano‐powders

Abstract

The potential of an organic monolith with incorporated titanium dioxide (TiO₂) and zirconium dioxide (ZrO₂) nanoparticles was evaluated for the selective enrichment of phosphorylated peptides from tryptic digests. A pipette tip was fitted with a monolith based on divinylbenzene (DVB) of highly porous structure, which allows sample to pass through the monolithic bed. The enrichment of phosphopeptides was enhanced by increasing the pipetting cycles during the sample preparation and a higher recovery could be achieved with adequate buffer systems. A complete automated process was developed for enrichment of phosphopeptides leading to high reproducibility and resulting in a robust method designed to minimize analytical variance while providing high sensitivity at high sample throughput. The effect of particle size on the selectivity of phosphopeptides was investigated by comparative studies with nano- and microscale TiO₂ and ZrO₂ powders. Eleven phosphopeptides from α-casein digest could be recovered by an optimized mixture of microscale TiO₂/ZrO₂ particles, whereas nine additional phosphopeptides could be retained by the same mixture of nano-structured material. When compared to conventional immobilized metal-ion affinity chromatography and commercial phosphorylation-enrichment kits, higher selectivity was observed in case of self fabricated tips. About 20 phosphopeptides could be retained from α-casein and five from β-casein digests by using TiO₂ and ZrO₂ based extraction tips. Further selectivity for phosphopeptides was demonstrated by enriching a digest of in vitro phosphorylated extracellular signal regulated kinase 1 (ERK1). Two phosphorylated peptides of ERK1 could be identified by MALDI-MS/MS measurements and a following MASCOT database search.