RESUMO
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/âinhibition 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 âMB â468 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-âMB-â468 cells.
Assuntos
Espectrometria de Massas/métodos , Proteínas de Membrana/análise , Fosfoproteínas/análise , Proteoma , Linhagem Celular Tumoral , Humanos , Fosforilação , Isoformas de Proteínas , Splicing de RNA , Transdução de SinaisRESUMO
Cancers are genetically and clinically very heterogeneous diseases; therefore, various proteomic studies have been trying to find bio-markers which can facilitate prognosis, diagnosis or treatment of these oncological diseases. The mass spectrometry is an effective tool for identification, quantitation, and characterization of biomolecules in the complex bio-logical samples. The first step suitable for selection of bio-markers called discovery proteomics provides a detailed analysis of the samples contributing to the identification of proteins, comparison of their presence in the samples, and selection of the convenient candidates for the prospective bio-markers. The next step of proteomics analysis is directed towards verification of chosen bio-markers with the approach called targeted proteomics. This technique evaluates presence and quantity of the proteins (biomarkers) in clinically precisely defined samples. This article focuses on the description of various approaches suitable for the quantitative analysis of the proteins connected with mass spectrometry.
Assuntos
Espectrometria de Massas/métodos , Neoplasias/química , Proteínas/análise , Biomarcadores/análise , Humanos , ProteômicaRESUMO
Recently, mass spectrometry has become a powerful tool in cancer research. Mass spectrometry represents the method that allows identification, quantification and characterization of proteins in bio-logical samples. Nowadays, it is mainly used for bio-marker discovery that can enable early detection of cancer. This article is focused on protein analysis by mass spectrometry. At first, mass spectrometry and its importance in proteomics are described. Subsequently ionization type and mass analyzers are discussed. This relates to the possibility of online or offline analysis connection with separation techniques, such as liquid chromatography and electrophoresis. Different approaches for preparing proteins and methods of analysis of bio-molecules using mass spectrometers are described. In addition, the possibility of mass spectrometric analyses of samples and data processing are discussed.