Application Of Gel-based Proteomic Approachs In Prostate Cancer Research

Gel-based proteomics generates qualitative and quantitative protein behavioral data, and as such it plays a central role in proteomic studies, particularly in the analysis of clinical biopsy specimens. In this study, efforts focused on 2DE, protein visualization, and protein in-gel digestion were made to ensure success of gel-based proteomics. With the optimized techniques, I conducted the proteomic analysis of prostate cancer using prostate needle biopsy (PNBX) specimens. This study not only establishd reliable gel-based proteomic approaches for various biological samples, but also defined the proteomic features implicated in PNBX specimens, and profiled the candidate biomarkers of prostate cancer.In the part of methodology development, first, comparisons referring to resolution and reproduction were made between two 2DE techniques, ISO-DALT and IPG-DALT, and the later was selected for its better results. To address the horizontal streaks in the alkaline region, four strategies were introduced and compared including elevating the concentration of DTT, shortening IEF duration, using the reducing agent TCEP and alkylating agent 4-VP in sample preparation, and anodic cup-loading. Thereafter an IPG-DALT separation system suiting to analyze the protein composition of biofluids (serum and urine), cells, or tissue was established. Second, using BSA and 293T cell proteins as biological models, I compared seven widely used protein visualization methods: Neuhoff CCB, Blue silver and five popular silver stains (LKB SN, He SN, Yan SN, Vorum SN, and Blum SN) with respect to sensitivity, background, dynamic range, and MALDI-TOF MS compatibility. With the rusults, I not only obtained the full-scale information about the stain efficiency and MS compatibility of these seven methods, but also got insight into the mechanism of the protein staining. Yan SN was selected in analytical gels for its good stain efficiency and acceptable MALDI-TOF MS compatibility, and Neuhoff CCB was selected in semi-preparative gels for its excellent MS compatibility and good conformability with Yan SN. Last, to improve the MS identification, studies were performed to determine the compatibility of in-gel trypsin digestion of stained protein spots with MS, to analyze the effects of reduction and alkylation on 2DE spot identification, and to evaluate time necessary for in-gel trypsin digestion for protein characterization with MS.Furthermore, the optimized gel-based proteomic approaches were used to analyze the PNBX specimens. Prostate cancer (PCa) is a significant cause of the morbidity and mortality worldwide. PNBX is a specficial clinical material obtained in the diagnose stage. Proteomic analysis of PNBX specimens is critical to our understanding of human prostate cancer. Twenty-six PNBX specimens from patients with PCa or benign prostatic hyperplasia (BPH) were collected. It is well-known that PNBX have false-negative rates approaching 30%, depending on the technique used. So protein samples were then subjected to immunoblot analysis for the malignant marker, AMACR, to test the tissue homogeneity. 14 BPH specimens and 9 AMACR-positive PCa specimens were further subjected to comparative proteomic analysis. 2DE revealed that 52 protein spots exhibited statistically significantly changes among PCa and BPH groups. Total 228 spots were analyzed further by MALDI-TOF/TOF MS. The two most notable groups of proteins identified included latent androgen receptor co-regulators [FLNA(7-15) and FKBP4] and enzymes involved in mitochondrial fatty acidβ-oxidation (ECHS1 and DCI). FLNA(7-15), FKBP4, ECHS1, DCI, CCT6A, and MFAP4 exhibited consistent expression patterns and may be useful for the development of diagnostic markers. An imbalance in the expression of peroxiredoxin subtypes was noted in PCa specimens. Different post-translationally modified isoforms of HSP27 and HSP70.1 were identified, and certain modifications to them were specifically linded to PCa. Furthermore, PSA and ACPP, which were previous defined as PCa markers, lacked significant change between PCa and BPH PNBX specimens. Importantly, changes in FLNA(7-15), FKBP4, and PRDX4 expression were confirmed by immunoblot analysis. The results suggest that the gel-based proteomic approach is useful for developing a more complete picture of the protein profile of PNBX specimen. The proteins identified by this approach may be useful molecular targets for PCa diagnostics and therapeutics.