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 (TiO(2)) and zirconium dioxide (ZrO(2)) 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 TiO(2) and ZrO(2) powders. Eleven phosphopeptides from alpha-casein digest could be recovered by an optimized mixture of microscale TiO(2)/ZrO(2) 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 alpha-casein and five from beta-casein digests by using TiO(2) and ZrO(2) 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.

Polyhedral oligomeric silsesquioxanes as functional monomer to prepare hybrid monolithic columns for capillary electrochromatography and capillary liquid chromatography

“…Nanoparticles were recently explored to functionalize the monoliths with specific selectivity. The approaches are divided into two categories: (i) direct encapsulation of nanoparticles into the monolith in the process of polymerization [21][22][23] and (ii) modification of surface with nanoparticles after monolith formation [24][25][26][27][28][29][30]. The latter approach results in a remarkable increase in the effective surface area of monolith, which was successfully applied for CEC and cLC separation, and solid-phase extraction as well.…”

Binder-Free TiO2 Monolith-Packed Pipette Tips for the Enrichment of Phosphorylated Peptides

“…The use of polymer in tip packing reduces the content of functional TiO 2 ; moreover, the polymer matrix may partially cover the TiO 2 surface and thus reduce the effective surface area for p-peptide binding. Compared with the binder (polymer)-assisted tip in the current study and previous reports, [12] the binder-free T-tip shows a lower detection limit because there is a higher availability of functional groups on the surface of the anatase in the absence of surface-bound binder, which enables the sensitive detection of p-peptides in a trace amount.…”

“…S3, Supplementary Material), in accordance with a previous report. [12] Considering the ERK level in biological samples is usually very low, tips with sufficient enrichment ability at low concentrations are essential. The enrichment test was further conducted with two lower concentrations, 50 (Fig.…”

“…The use of a functional tip in pretreatment is beneficial for minimizing sample loss, simplifying the operation procedures, and reducing the contamination risk. However, the packing of TiO 2 into the tip in literature reports is generally assisted by polymers for adhesion [10,12] or a plug [8] to prevent materials from leaking. The mix of adhesive polymer would reduce the functional TiO 2 content, which may result in a decrease in enrichment efficiency, and the use of a plug makes the packing procedure complicated.…”

“…[9] TiO 2 and other metal oxides have been packed in pipette tips as a convenient and frequently used device for in situ sample pretreatment. [8,[10][11][12] There are commercial pipette tips on the market (e.g. NuTip, Titansphere Phos-TiO Kit) employing embedded TiO 2 for p-peptide and protein enrichment.…”