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Gaithersburg, MD, United States

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.


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.


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.


Grant
Agency: Department of Defense | Branch: Defense Advanced Research Projects Agency | Program: SBIR | Phase: Phase II | Award Amount: 370.88K | Year: 2003

This proposal describes an integrated research and development plan leading to a novel portable biowarfare detection platform capable of meeting all key requirements for universal pathogen detection. Effective technologies for the detection andidentification of biological warfare agents (BWAs) are of critical and growing importance. Despite ongoing advances in this area, current technologies are limited by a combination of poor sensitivity, excessive reagent usage, lengthy detection cycles,excessive power consumption, and poor BWA discrimination. In contrast, the technology proposed here, called MS3, is capable of identifying a wide range of BWAs including bacteria, toxins, and viruses by using a novel microfluidic detection platform basedon protein biomarkers. Furthermore, the platform will provide high throughput and high sensitivity, with significant reductions in false positives. It is inherently low power and portable for field-deployable applications, but compatible with backendanalysis methods to validate field measurements as needed. The MS3 system requires no bioreagents for improved simplicity, repeatability, and overall robustness. To meet the demands from a range of potential markets, the overall system will be low-cost,with disposable detection elements to eliminate the danger of cross-contamination without the need for complex and time-consuming system regeneration.

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