Acetonitrile-assisted enzymatic digestion can facilitate the bottom-up identification of proteins of cancer origin.

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.

Consequences of the natural retinoid/retinoid X receptor ligands action in human breast cancer MDA-MB-231 cell line: Focus on functional proteomics.

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.