| Because human prostate plays important roles in men's health, there is a great need to develop sensitive bioanalytical methods to examine the protein machinery in the prostate, in order to characterize disease marker proteins and other applications. The researches presented focus on comprehensive analyses of the proteome/phosphoproteome in human prostate tissue by a combination of proteomics technologies, including a novel in-gel IEF-LC-MS/MS methodology.; In the first project, the proteome in human control prostate tissue was analyzed with 2D-PAGE, LC-MS/MS and database search. A 2D reference map was constructed and 101 protein spots were identified, representing 86 different proteins. The data serve as a reference for our on-going comparative proteomics studies, and they will be uploaded onto UT proteomics website.; In the second project, the novel in-gel IEF-LC-MS/MS was applied as an alternative platform to complement the limitations of 2D-PAGE. The proteins were separated by IEF on an IPG strip. The strip was cut into 50 sections. The proteins in each section were digested, and the peptide mixtures were analyzed with LC-MS/MS. After database search, 836 proteins were identified. Compared to 2D-PAGE-based method, more sample loading and higher throughput were achieved; more hydrophobic proteins and proteins with extreme pI and MW values were identified. The data represent one of the largest data sets of the human prostate proteome to date.; In the third project, the phosphoproteome in human cancer prostate tissue was analyzed with non-gel-LC-MS/MS and in-gel IEF-LC-MS/MS: The extracted proteins were either digested in solution or in-gel following IEF. IMAC columns were used to enrich the phosphopeptides. Ziptip C18 SPE was conducted, followed by LC-MS/MS analyses. After database search and spectra inspection, 24 phosphorylation sites were identified from non-gel-based method and 19 of them were reproducibly identified. The in-gel IEF-based method showed better results because the separation decreased the complexity of the sample: 62 phosphorylation sites were found.; These findings bring new knowledge on the molecular characteristics of the prostate proteins, which will be of value for future studies in the group as well as for other members of the scientific community who are engaged in prostate cancer research. |
