The main objective of this study was to develop an effective in-gel trypsin digestion protocol using aqueous-acetonitrile solvent system to facilitate MS analysis and maximize the number of identified proteins from biological samples. The procedure, where 80% acetonitrile was present in the trypsin reaction mixture, increased the number of matched peptides, and allowed the identification of more proteins with higher coverage than the common digestion protocol. Vimentin, annexins, tubulin, actin, peptidyl-prolyl cis-trans isomerase or alpha-enolase are examples of important proteins that change during the progress cancer. These were isolated from human breast cancer cells and were used for this study.
- MeSH
- acetonitrily chemie MeSH
- lidé MeSH
- nádorové buněčné linie MeSH
- nádorové proteiny analýza izolace a purifikace metabolismus MeSH
- nádory metabolismus patologie MeSH
- peptidy analýza MeSH
- proteomika metody MeSH
- spektrometrie hmotnostní - ionizace laserem za účasti matrice MeSH
- trypsin metabolismus MeSH
- vysokoúčinná kapalinová chromatografie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The main intention of this study was the investigation of impact of natural biologically active ligands of nuclear retinoid/retinoid X receptors (all-trans and 9-cis retinoic acid) on proteomic pattern in human estrogen receptor negative breast cancer cell line MDA-MB-231. For this purpose, proteomic strategies based on bottom-up method were applied. The total cell proteins were extracted utilizing a commercially Radio-Immunoprecipitation Assay (RIPA) buffer and separated on 2D sodium dodecyl sulfate polyacrylamide gel electrophoresis (2D SDS-PAGE). The proteins were subsequently digested in-gel by trypsin and their characterization was achieved by MALDI-TOF/TOF. By employing PDQuest™ software, we identified more than 50 proteins affected by retinoic acid isomers. For more information, 9 proteins which are associated with tumor process were selected. We determined that derivatives of retinoic acid led to significantly reduced level of proteins belonging to metabolic pathway (e.g. glyceraldehyde-3-phosphate dehydrogenase or pyruvate kinase 2) or to other cellular processes as apoptosis, regulation of transcription process or epithelial-mesenchymal transition (e.g. annexins, nucleoside diphosphate kinase B, vimentin). On the other hand all-trans retinoic acid treatment indicates up-regulated effect for heterogeneous nuclear ribonucleoprotein A2/B1.
- MeSH
- antitumorózní látky farmakologie MeSH
- apoptóza účinky léků MeSH
- elektroforéza v polyakrylamidovém gelu MeSH
- epitelo-mezenchymální tranzice účinky léků MeSH
- heterogenní jaderné ribonukleoproteiny skupiny A-B genetika metabolismus MeSH
- lidé MeSH
- ligandy MeSH
- nádorové buněčné linie MeSH
- nádory prsu genetika MeSH
- proteomika * MeSH
- regulace genové exprese u nádorů * MeSH
- retinoidní X receptory genetika metabolismus MeSH
- tretinoin farmakologie MeSH
- upregulace MeSH
- Check Tag
- lidé MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- Publikační typ
- abstrakt z konference MeSH
Retinoic acid (all-trans and 9-cis) isomers represent important therapeutic agents for many types of cancers, including human breast cancer. Changes in protein composition of the MCF-7 human breast cancer cells were induced by all-trans retinoic acid, 9-cis retinoic acid, and their combination and subsequently proteomic strategies based on bottom-up method were applied. Proposed approach was used for the analysis of proteins extracted from MCF-7 human breast cancer cell line utilizing a commercially manufactured kit RIPA and separated on two dimensional (2D) sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) after treatment with both retinoic acid isomers. We found significant differences in occurrence of proteins probably affecting the cell migration process in tumour cells. Heat shock protein 27, ribonucleoprotein SmD3, and cofilin-1 were significantly upregulated after treatment with combination of individual retinoic acid isomers. On the other hand, AP-5 complex subunit beta-1 shows the different response. Thus, the results might help to find the answer to important medical questions on (i) the identification of signaling pathways affected by retinoic acid isomers or (ii) how the observed proteomic pattern might reflect the effectiveness of retinoic acids treatment.
- MeSH
- 2D gelová elektroforéza MeSH
- adaptorové proteiny vezikulární transportní metabolismus MeSH
- cytoskelet účinky léků metabolismus MeSH
- elektroforéza v polyakrylamidovém gelu MeSH
- invazivní růst nádoru MeSH
- jádro snRNP - proteiny metabolismus MeSH
- kofilin 1 metabolismus MeSH
- lidé MeSH
- nádorové proteiny metabolismus MeSH
- nádory prsu farmakoterapie metabolismus patologie MeSH
- pohyb buněk účinky léků MeSH
- proteiny tepelného šoku HSP27 metabolismus MeSH
- proteomika * metody MeSH
- protokoly antitumorózní kombinované chemoterapie farmakologie MeSH
- tretinoin farmakologie MeSH
- Check Tag
- lidé MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
While a plant cell wall is formed by a complex of various components, including polysaccharides and structural proteins, its composition and representation may vary during cell growth. Currently, plant research targets the proteins participating in wall loosening. Multiple classes of enzymes, including various hemicellulases and cellulases, are required for plant material degradation to achieve the maximum decomposition. Identifying the set of proteins involved in the breakdown of cell-wall polymers is important to understand plant material conversion into suitable products. The objective of this study was to describe a method which can be used to carry out proteomics analysis of complex plant samples and identify enzymes degrading biomass. For this purpose we used proteomic techniques including gel electrophoresis, high pressure liquid chromatography combinated with mass spectrometry followed by data evaluation using databases searching. Results show that more than 50 % of these activities correspond to enzymes with proteolytic function. This study was focused primarily on enzymes able to breakdown the lignocellulosic and hemicellulosic parts that are very important for the material conversion into required products of degradation.
- MeSH
- angiografie využití MeSH
- nádory ledvin diagnóza MeSH
- Publikační typ
- kongresy MeSH
