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Analysis of protein phosphorylation by monolithic extraction columns based on poly(divinylbenzene) containing embedded titanium dioxide and zirconium dioxide nano‐powders
Author(s) -
Rainer Matthias,
Sonderegger Harald,
Bakry Rania,
Huck Christian W.,
Morandell Sandra,
Huber Lukas A.,
Gjerde Douglas T.,
Bonn Günther K.
Publication year - 2008
Publication title -
proteomics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.26
H-Index - 167
eISSN - 1615-9861
pISSN - 1615-9853
DOI - 10.1002/pmic.200800448
Subject(s) - monolith , chromatography , selectivity , phosphopeptide , divinylbenzene , titanium dioxide , zirconium , chemistry , extraction (chemistry) , microscale chemistry , sample preparation , materials science , inorganic chemistry , polymer , peptide , organic chemistry , biochemistry , styrene , mathematics , copolymer , metallurgy , catalysis , mathematics education
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 α‐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 α‐casein and five from β‐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.