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New highly selective interface for phosphoproteome profiling

Prof. Zuzana Bílková, University of Pardubice; Dr Jan Macák, University of Pardubice; Rudolf Kupčik, University of Pardubice; Dr Pavel Řehulka, University of Pardubice

University of Pardubice


Phosphorylation is a reversible covalent modification of proteins/peptides which alters the functional activity of affected proteins / peptides and influences many processes taking place in cells, tissues or body. Phosphoproteomics as the relatively new field of science focuses on studying these processes as well as quantifying the dynamic changes of phosphorylated proteins over the time to identify and describe the relationship of the regulation of phosphorylation with the pathogenesis and development of serious diseases, for example Parkinson´s disease, where phosphorylation is involved in neurodegenerative mechanisms of its pathology or Alzheimer´s disease where Tau hyperphosphorylation is a common example of phosphorylation related pathology. To have a qualified look at the phosphorylation biological processes dependent on signaling networks a comprehensive analysis of the phosphorylated proteins is essential. However, the low abundance of phosphopeptides, their low ionization efficiency, labile phosphate moiety and the interference of non-phosphorylated peptides strongly limit the comprehensive characterization of protein phosphorylation and can lead to results distortion or misleading conclusions. Although many materials with declared affinity for phosphorylated molecules have already been developed and applied in phosphoproteome analysis (profiling), none of them addresses all of the aforementioned aspects at the same time.


It is undeniable that specific phosphopeptides enrichment is critical for phosphoproteomic analysis. To address needs for phosphopeptides enrichment approach such as high selectivity and simplicity of operation, a high surface area interface, based on anodic one-dimensional (1D) TiO2 nanotubes homogeneously decorated by Fe3O4 nanoparticles (TiO2NTs@Fe3O4NPs) was introduced. The combination of TiO2NTs@Fe3O4NPs with tailored isolation protocol offers an enhanced separation specificity, enrichment ability and possibility to widen the range of identified peptides compared to the established TiO2 microspheres and IMAC-based protocols. Presented material (TiO2NTs@Fe3O4NPs) possesses further unique advantages: it is nontoxic, robust, it can be easily separated from any solution due to its intrinsic magnetism, and being based on TiO2 it can be simply decontaminated by UV-light-induced photocatalytic treatment at low costs and reused again at the same quality. Our results have proved that the material (TiO2NTs@Fe3O4NPs) retains almost exclusively phosphopeptides and shows 50% better preferential affinity for double or triple phosphorylated peptides. Moreover it retains a high number of unique phosphopeptides impossible to be isolated by other techniques. This attribute is fully exploitable mainly for analysis of hyperphosphorylated proteins associated with serious degenerative diseases.

Commercial Opportunity

The invention is offered for co-development and licensing. Unique properties of TiO2NTs@Fe3O4NP may open new pathways for the isolation and identification of clinically important biomolecules and for a whole range of in vitro life science applications.

Development Status

Series of samples were prepared. Their ability to separate selected biomolecules was proven at analytical (trace amount) scale. These materials can be easily up-scaled for preparative use.

Patent Situation

US patent application pending, priority 2014. CZ patent granted. Filing of a complementary priority patent application is planned for February 2018.

Further Reading

Paper recently submitted to Journal of Chromatography A (January 2018).