title: [Analysis of Phosphoproteome Changes in MDA MB 468 Cancer Cell Line in Response to Expression of p63 Isoforms Using Mass Spectrometry].
abstract: 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 wildtype 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.
authors.forename: P
authors.initials: P
authors.lastname: Dvořáková
authors.forename: M
authors.initials: M
authors.lastname: Nekulová
authors.forename: J
authors.initials: J
authors.lastname: Holčaková
authors.forename: B
authors.initials: B
authors.lastname: Vojtěšek
authors.forename: L
authors.initials: L
authors.lastname: Hernychova
mesh_terms: D045744:Cell Line, Tumor; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods*; D008565:Membrane Proteins / Q000032:analysis*; D010750:Phosphoproteins / Q000032:analysis*; D010766:Phosphorylation; D020033:Protein Isoforms; D020543:Proteome; D012326:RNA Splicing; D015398:Signal Transduction
chemical_list: C081876:CKAP4 protein, human; D008565:Membrane Proteins; D010750:Phosphoproteins; D020033:Protein Isoforms; D020543:Proteome