Analysis of protein phosphorylation by monolithic extraction columns based on poly(divinylbenzene) containing embedded titanium dioxide and zirconium dioxide nano-powders.
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.
Rainer
Matthias
M
Institute of Analytical Chemistry and Radiochemistry, Leopold-Franzens University, Innsbruck, Austria.
Sonderegger
Harald
H
Bakry
Rania
R
Huck
Christian W
CW
Morandell
Sandra
S
Huber
Lukas A
LA
Gjerde
Douglas T
DT
Bonn
Günther K
GK
D000818:Animals; D005982:Glutathione Transferase / Q000235:genetics; D007202:Indicators and Reagents; D019950:Mitogen-Activated Protein Kinase 1 / Q000032:analysis / Q000235:genetics; D048052:Mitogen-Activated Protein Kinase 3 / Q000032:analysis / Q000235:genetics; D053758:Nanoparticles; D010446:Peptide Fragments / Q000032:analysis; D010748:Phosphopeptides / Q000032:analysis*; D010766:Phosphorylation; D016062:Porosity; D011208:Powders; D011506:Proteins / Q000378:metabolism*; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000235:genetics; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013343:Styrenes; D053719:Tandem Mass Spectrometry; D014025:Titanium; D014357:Trypsin; D015040:Zirconium
D007202:Indicators and Reagents; D010446:Peptide Fragments; D010748:Phosphopeptides; D011208:Powders; D011506:Proteins; D011993:Recombinant Fusion Proteins; D013343:Styrenes; C009495:titanium dioxide; C056745:poly(divinylbenzene); D015040:Zirconium; D014025:Titanium; D005982:Glutathione Transferase; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D014357:Trypsin; C028541:zirconium oxide