Date of Award
Doctor of Philosophy (PhD)
Sarka Beranova-Giorgianni, Ph.D.
Dominic M. Desiderio, Ph.D. Ivan C. Gerling, Ph.D. Wei Li, Ph.D. Duane D. Miller, Ph.D.
In the current dissertation, we focused on the development and application of multiple mass spectrometry-based bioanalytical platforms for phosphoproteomic characterization in cell culture and clinical specimens of prostate cancer; and on the application of optimized methods to analysis of differential protein expression to reveal molecular mechanism of drug action in animal model of prostate cancer.
Characterization of the phosphoproteome in prostate cancer
Our study in phosphoprotein signatures on a large scale in prostate cancer focused on the LNCaP human prostate cancer cell line, and on human prostate cancer tissue.
For the LNCaP prostate cancer cell line, we applied a combination of analytical platforms: (1) a novel in-gel isoelectric focusing (IEF) LC-MS/MS analytical platform; (2) a 2-DE based platform combined with phospho-specific staining.
The in-gel IEF LC-MS/MS analytical methodology used in the study included separation of the LNCaP proteins by in-gel isoelectric focusing; digestion of the proteins with trypsin; enrichment of the digests for phosphopeptides with immobilized metal ion affinity chromatography (IMAC); analysis of the enriched digests by LC-MS/MS; and identification of the phosphorylated peptides/proteins through searches of the Swiss-Prot protein sequence database. With in-gel IEF based analytical platform, we have characterized over 600 different phosphorylation sites in 296 phosphoproteins in the LNCaP prostate cancer cell line. This panel of the LNCaP phosphoproteins was 3-fold larger than the panel obtained in our previous work, and is the largest phosphoprotein panel in prostate cancer reported to date. The phosphoproteins identified in this study belonged to various locations within the cell and were involved in various processes including cell differentiation, transcription regulation, and intercellular signal transduction.
We also developed a 2-DE based platform, in combination with multiplexed staining and LC-MS/MS, for the identification of LNCaP phosphoproteins. In this study, we applied 2-DE as separation technique, Pro-QTM Diamond stain as phosphoprotein detection method, LC-MS/MS and database searches for protein identification to investigate the phosphoproteins in the LNCaP prostate cancer cell line. Proteins identified from spots of interest were shown to be highly relevant to prostate cancer. We demonstrated the feasibility of using 2-DE with phospho-specific stain and mass spectrometry to investigate the phosphoproteins in the LNCaP cell line. This methodology complements the in-gel IEF LC-MS/MS platform that we used for phosphoproteomics study; it will be of particular value for future comparative studies of phosphoproteins in various physiological states.
For prostate cancer tissue, a gel-free approach was performed to analyze five prostate cancer tissue specimens to obtain phosphoproteomic signature of prostate cancer for biomarker discovery. Proteins were extracted with Trizol reagent, and then in-solution digested with trypsin. Phosphopeptides were enriched with IMAC, and analyzed the phosphorylated peptides/proteins by LC-MS/MS with identification through searches of the Swiss-Prot protein sequence database. The panels obtained for prostate cancer tissue contain 15-24 phosphoproteins. Some of the characterized phosphoproteins were present in all five specimens; in addition, each specimen also produced a unique set of phosphoproteins. The findings provided a direct glimpse into the phosphoprotein machinery operating within the human prostate cancer tissue. This pilot study focused on a small set of specimens. The phosphoprotein panels that were obtained contained a number of proteins that were unique to a particular specimen.
Comparative proteomics study of drug effects in prostate cancer
We carried out the first comparative proteomics study for the examination of the effects of bicalutamide/embelin combination treatment on prostate tumors by characterizing the alterations in protein expression that was induced upon treatment of mice bearing prostate tumors with anticancer combination therapy.
A comparative proteomic strategy based on 2-DE coupled with LC-MS/MS was performed on mouse prostate tumor tissue. Proteins from the mouse prostate tumors were extracted with Trizol, and the protein mixtures were separated by 2-DE. Differences in the protein profiles for the different treatment groups were evaluated by computer-assisted analysis of SYPRO Ruby stained 2-DE gels. LC-MS/MS and database searches were used to identify differentially expressed proteins. Pathway analysis was carried out on the dataset of identified proteins with the Ingenuity bioinformatics tool. Out of the 33 differentially expressed protein spots, 30 protein spots were identified and grouped into various functional classes. The major protein categories were metabolism (52%), stress response (12%), protein biosynthesis (13%) and apoptosis (11%), suggesting that alterations in these processes may be involved in the mechanism of drug action. Proteins associated with oxidative stress were up-regulated, which indicated that treatment with bicalutamide/embelin may affect the redox balance within the prostate tumor, and this effect may contribute to tumor suppression.
Chen, Li , "Application of Different Bioanalytical Workflows for Proteomics of Prostate Cancer" (2009). Theses and Dissertations (ETD). Paper 43. http://dx.doi.org/10.21007/etd.cghs.2009.0050.