University of PardubiceFaculty of Chemical Technology

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Kinases-superparamagnetic beads for hyperphosphorylation of peptides/proteins

Authors: Slováková Marcela | Hromádková Lenka | Kupčík Rudolf | Charvátová Andrea | Přikryl Petr | Jankovičová Barbora | Vajrychová Marie | Řípová Daniela | Bílková Zuzana

Year: 2015

Type of publication: ostatní - přednáška nebo poster

Page from-to: nestránkováno

Titles:

Language	Name	Abstract	Keywords
eng	Kinases-superparamagnetic beads for hyperphosphorylation of peptides/proteins	Hyperphosphorylated Tau protein preparation, as the marker of Alzheimer disease, was the purpose for the kinases immobilizations. We focused on the immobilization of proline-directed protein kinases: extracellular signal-regulated kinase (ERK2) and glycogen synthase kinase 3β (GSK-3β). Today, the carriers with immobilized enzymes as recoverable, stable and specific catalysts are routinely applied in many fields of industry and science, e.g. basic biochemical and pharmacological research. Magnetic microparticles carriers bring the advantage of the non-contaminating and very specific and sensitive reaction on their substrates, peptides and proteins. Magnetic microparticles were used for the kinase immobilization: BcMag®-Aldehyde, SeraMag-carboxyl, SIMAG-PGL, SIMAG-IDA-Co2+, and SIMAG-IDA-Ni2+. Relevant methods of covalent immobilization, non-oriented and oriented, were chosen and reaction conditions were optimized. Phosphorylations of low molecular substrates followed by MALDI MS analysis were used as detection system. Operational and storage stabilities of kinase-superparamagnetic beads were observed. The both soluble and immobilized ERK2 and GSK-3β were applied for recombinant tau protein phosphorylation. Tryptic phosphopeptides enrichment was performed by ion-metal affinity chromathography using TiO2 magnetic nanoparticles. The level and the position of phosphorylation sites were identified by using MALDI-LTQ-Orbitrap MS. Present results document very well the ability of ERK2- and GSK-3-superparamagnetic beads to phosphorylate the target peptides or proteins with desired efficiency and specificity. The process of phosphorylation can be better controlled; subsequent purification of phosphorylated Tau can be omitted.