CEEPC/IPM/CMSC - Abstrakt prezentace

(Česká konference hmotnostní spektrometrie 2019 - ThS-013)
Application of two amorphous TiO2 nanotubes-based materials for highly selective phosphopeptide enrichment followed by nanoLC-MS/MS analysis

Rudolf Kupcik 1, Pavel Rehulka 2 *, Jan M. Macak 3,4, Helena Rehulkova 2, Hanna Sopha 3,4, Ivo Fabrik 2, Jana Klimentova 2, Zuzana Bilkova 1

  1. Department of Biological and Biochemical Sciences, University of Pardubice, Pardubice
  2. Department of Molecular Pathology and Biology, University of Defence, Hradec Kralove
  3. Center of Materials and Nanotechnologies, University of Pardubice, Pardubice
  4. Central European Institute of Technology, Brno University of Technology, Brno

Abstrakt

Phosphorylation is a reversible covalent modification and plays a critical role in many biological processes. Mass spectrometry as an important tool for phosphoproteome analysis needs preceding phosphopeptide enrichment step for analysis of complex peptide mixtures due to low abundance and low ionization efficiency of phosphopeptides. Here we demonstrate the enrichment of phosphopeptides with substantially higher selectivity by introducing of metal oxides-based nanotubes. Two developed nanomaterials, the amorphous TiO2 nanotubes (TiO2NTs) and the same material decorated by Fe3O4 nanoparticles (TiO2NTs@Fe3O4NPs) with superparamagnetic properties, were tested for phosphopeptides enrichment and compared with commercial TiO2 microspheres. To compare the aforementioned materials, digest of BSA and alpha-casein as a simple (phospho)peptide mixture and the whole cell lysate digest of Jurkat T-cells representing a real complex peptide sample were tested for phosphopeptide enrichment. The simple mixture was analyzed with MALDI-IT-Orbitrap MS and the cell lysate digest was measured using nanoLC-MS/MS (Q-Exactive). The obtained results revealed that TiO2NTs and TiO2NTs@Fe3O4NPs used for phosphopeptides enrichment from complex biological samples perform similarly as TiO2 microspheres in the number of identified phosphopeptides. However, the presented nanomaterials showed a significantly enhanced enrichment selectivity which enables reduction of ionization suppression of phosphopeptides within nanoLC-MS analysis. The numbers of identified non-phosphorylated peptides were approximately 4-fold lower as compared to TiO2 microspheres. Moreover, further modifications in the enrichment protocol led to increase in the overall phosphoproteome coverage of the cell lysate digest.

* Korespondující autor: pavel.rehulka@unob.cz

Poděkování:

This work was supported from European Regional Development Fund-Project Strengthening interdisciplinary cooperation in research of nanomaterials and their effects on living organisms_No. CZ.02.1.01/0.0/0.0/17_048/0007421, European Research Council (project nr. 638857), Ministry of Education, Youth and Sports of the Czech Republic (projects nr. LM2015082, LQ1601 and CZ.02.1.01/0.0/0.0/16_013/0001829) and a long-term organization development plan from the Faculty of Military Health Sciences, University of Defence (Czech Republic).


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