Entity

Time filter

Source Type

Gaithersburg, MD, United States

Jinawath N.,Johns Hopkins Medical Institutions | Jinawath N.,Mahidol University | Vasoontara C.,Johns Hopkins Medical Institutions | Jinawath A.,Mahidol University | And 10 more authors.
PLoS ONE | Year: 2010

Background: Ovarian cancer is one of the most lethal types of female malignancy. Although most patients are initially responsive to platinum-based chemotherapy, almost all develop recurrent chemoresistant tumors and succumb to their diseases. Elucidating the pathogenesis underlying drug resistance is fundamental to the development of new therapeutics, leading to improved clinical outcomes in these patients. Methods and Findings: We compared the proteomes of paired primary and recurrent post-chemotherapy ovarian highgrade serous carcinomas from nine ovarian cancer patients using CIEF/Nano-RPLC coupled with ESI-Tandem MS. As compared to their primary tumors, more than half of the recurrent tumors expressed higher levels of several proteins including CP, FN1, SYK, CD97, AIF1, WNK1, SERPINA3, APOD, URP2, STAT5B and RELA (NF-kB p65), which were also validated by quantitative RT-PCR. Based on shRNA screening for the upregulated genes in in vitro carboplatin-resistant cells, we found that simultaneous knockdown of RELA and STAT5B was most effective in sensitizing tumor cells for carboplatin treatment. Similarly, the NF-kB inhibitor, BMS-345541, and the STAT5 inhibitor, Dasatinib, significantly enhanced cell sensitivity to carboplatin. Moreover, both RELA and STAT5 are known to bind to the promoter region of Bcl-X, regulating its promoter activity. In this regard, augmented Bcl-xL expression was detected in carboplatin-resistant cells. Combined ectopic expression of RELA and STAT5B enhanced Bcl-xL promoter activity while treatment with BMS-345541 and Dasatinib decreased it. Chromatin immunoprecipitation of the Bcl-X promoter region using a STAT5 antibody showed induction of RELA and STAT5 DNA-binding segments both in nai{dotless}̈ve cells treated with a high concentration of carboplatin as well as in carboplatin-resistant cells. Conclusions: Proteomic analysis identified RELA and STAT5 as two major proteins associated with carboplatin resistance in ovarian tumors. Our results further showed that NF-kB and STAT5 inhibitor could sensitize carboplatin-resistant cells and suggest that such inhibitors can be used to benefit patients with carboplatin-resistant recurrent ovarian cancer. © 2010 Jinawath et al. Source


Wang C.,University of Maryland University College | Fang X.,Calibrant Biosystems | Lee C.S.,University of Maryland University College
Methods in Molecular Biology | Year: 2013

Due to the inherent disadvantage of biomarker dilution in complex biological fluids such as serum/plasma, urine, and saliva, investigative studies directed at tissues obtained from the primary site of pathology probably afford the best opportunity for the discovery of disease biomarkers. Still, the large variation of protein relative abundances with clinical specimens often exceeds the dynamic range of currently available proteomic techniques. Furthermore, since the sizes of human tissue biopsies are becoming significantly smaller due to the advent of minimally invasive methods and early detection and treatment of lesions, a more effective discovery-based proteomic technology is critically needed to enable comprehensive and comparative studies of protein profiles that will have diagnostic and therapeutic relevance. This review therefore focuses on the most recent advances in capillary electrophoresis-based single and multidimensional separations coupled with mass spectrometry for performing comprehensive proteomic analysis of clinical specimens. In addition to protein identification, monitoring quantitative changes in protein expression is essential for the discovery of disease-associated biomarkers. Comparative proteomics involving measurements in changes of biological pathways or functional processes are further expected to provide relevant markers and networks, molecular relationships among different stages of disease, and molecular mechanisms that drive the progression of disease. © Springer Science+Business Media, LLC 2013. Source


Fang X.,Calibrant Biosystems
Methods in molecular biology (Clifton, N.J.) | Year: 2013

Besides proteome complexity, the greatest bioanalytical challenge facing comprehensive proteomic analysis, particularly in the identification of low abundance proteins, is related to the large variation of protein relative abundances. In contrast to universally enriching all analytes by a similar degree, the result of the capillary isotachophoresis (CITP) stacking process is that major components may be diluted, but trace compounds are concentrated. Such selective enhancement toward low abundance proteins drastically reduces the range of relative protein abundances within complex proteomes and greatly enhances the resulting proteome coverage. Furthermore, CITP offers seamless combination with nano-reversed phase liquid chromatography (nano-RPLC) as two highly resolving and completely orthogonal separation techniques critically needed for analyzing complex proteomes. Source


Grant
Agency: Department of Defense | Branch: Army | Program: SBIR | Phase: Phase I | Award Amount: 120.00K | Year: 2004

This proposal aims to develop and validate a multidimensional protein separation platform based on polymer microfluidics technology which will be capable of mapping a wide range of post-translational modifications (PTMs) in complex protein samples. The proposed system employs three separation dimensions, namely: IEF, SDS gel electrophoresis, and MALDI-MS. The platform will combine IEF and SDS gel electrophoresis on a single microfluidic chip, with chip-level laser-induced fluorescence detection to record the separated protein positions, and an integrated electrospray tip array to elute separated protein bands from the chip onto a MALDI target plate for MALDI-MS analysis. On-target proteolytic digestion will be performed using two different enzymes to ensure a high degree of peptide coverage during MS analysis. This detection platform will eliminate the manually-intensive process of running standard 2-D gels, while also eliminating the sample dilution associated with traditional 2-D PAGE / MALDI-MS interfacing. Furthermore, the system is expected to provide complete 3-D separations in ~1 hour, approximately an order of magnitude faster than traditional 2-D PAGE / MALDI-MS, with greatly improved PTM analysis capabilities in a miniaturized format.


Grant
Agency: Department of Defense | Branch: Army | Program: SBIR | Phase: Phase II | Award Amount: 741.72K | Year: 2004

This Phase II SBIR proposal aims to develop a multidimensional protein separation platform based on polymer microfluidics technology which will be capable of mapping a wide range of post-translational modifications (PTMs) in complex protein samples. The proposed system employs three separation dimensions, namely: IEF, SDS gel electrophoresis, and MALDI-MS. The platform will combine IEF and SDS gel electrophoresis on a single microfluidic chip, with chip-level laser-induced fluorescence detection to record the separated protein positions, and an integrated electrospray ionization tip array to elute separated proteins from the chip onto a MALDI target for MALDI-MS analysis. This detection platform will eliminate the manually-intensive process of running standard 2-D gels, while also eliminating sample dilution associated with traditional 2-D PAGE / MALDI-MS interfacing. Furthermore, the system is designed to provide complete 2-D separations in under 15 min, and full PTM identification via MALDI-MS in under 1 hr, approximately an order of magnitude faster than traditional 2-D PAGE, with greatly improved PTM analysis capabilities in a miniaturized format.

Discover hidden collaborations