Analysis of Phosphoproteome Changes in MDA‑ MB‑ 468 Cancer Cell Line in Response to Expression of p63 Isoforms Using Mass Spectrometry

Česká verzia

Authors: P. Dvořáková; M. Nekulová; J. Holčáková; B. Vojtěšek; L. Hernychová
Authors place of work: Regionální centrum aplikované molekulární onkologie, Masarykův onkologický ústav, Brno
Published in the journal: Klin Onkol 2015; 28(Supplementum 2): 11-19
doi: https://doi.org/10.14735/amko20152S11

Summary

Compared to normal cells, tumor cells can show different activity of kinases and phosphatases resulting in altered phosphorylation states of proteins affecting their activity within various signaling pathways. The detection of these alterations is essential for development of targeted therapy based on activation/ inhibition of specific signaling pathways. Various methods can be used for detection of protein phosphorylation; however, a comprehensive assessment of phosphoproteome is performed by mass spectrometry. The differences in phosphoproteome were studied using MDA MB 468 cell line (with incorporated genes encoding isoforms of p63) derived from breast carcinoma. Cells with tetracycline‑induced expression of the p63 isoforms were compared to control cells with wild‑type expression. Denatured proteins from cell lysates were digested to peptides, enriched for phosphopeptides and subsequently separated using liquid chromatograph coupled with mass spectrometer Orbitrap Elite. Three different mass spectrometric methods were used for each sample analysis to find the most suitable conditions for the detection of phosphorylated peptides. Then phosphoproteins were identified and quantified. The number of identified phosphoproteins using all chosen mass spectrometric methods was similar; however, each method showed several unique phosphorylated proteins. Our analysis revealed that both p63 isoforms (TAp63α a ∆Np63α) mainly affected phosphorylation of proteins associated with RNA splicing in MDA MB 468 cells.

Key words:
mass spectrometry – phosphoproteins – signaling pathways – p63 isoforms – breast cancer

This study was supported by the European Regional Development Fund and the State Budget of the Czech Republic (RECAMO, CZ.1.05/2.1.00/03.0101), by the project MEYS – NPS I – LO1413, by MH CZ – DRO (MMCI, 002 09805), by IGA NT/14602-3/2013 and BBMRI_CZ (LM2010004).

The authors declare they have no potential conflicts of interest concerning drugs, products, or services used in the study.

The Editorial Board declares that the manuscript met the ICMJE “uniform requirements” for biomedical papers.

Submitted:
9. 4. 2015

Accepted:
20. 7. 2015

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