Calibrant Biosystems

Gaithersburg, MD, United States

Calibrant Biosystems

Gaithersburg, MD, United States
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Tsao C.-W.,National Central University | Tao S.,Calibrant Biosystems | Chen C.-F.,University of Maryland University College | Liu J.,University of Maryland University College | Devoe D.L.,University of Maryland University College
Microfluidics and Nanofluidics | Year: 2010

We developed a method of interfacing microfluidics with mass spectrometry (MS) using a robotic spotting system to automate the contact spotting process. We demonstrate that direct and automated spotting of analyte from multichannel microfluidic chips to a custom microstructured MALDI target plate was a simple, robust, and high-throughput method for interfacing parallel microchannels using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). Using thermoplastic cyclic olefin copolymer (COC) polymer microfluidic chips containing eight parallel 100 μm × 46 μm microchannels connected to a single input port, spotting volume repeatability and MALDI-MS signal uniformity are evaluated for a panel of sample peptides. The COC microfluidic chips were fabricated by hot embossing and solvent bonding techniques followed by chip dicing to create open ends for MS interfacing. Using the automatic robotic spotting approach, microfluidic chip-based reversed-phase liquid chromatography (RPLC) separations were interfaced with electrochemically etched nanofilament silicon (nSi) target substrate, demonstrating the potential of this approach toward chip-based microfluidic separation coupled with matrix-free laser desorption/ionization mass spectrometry. © 2009 Springer-Verlag.


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.


Lu J.,U.S. National Institutes of Health | Ksendzovsky A.,U.S. National Institutes of Health | Yang C.,U.S. National Institutes of Health | Mehta G.U.,U.S. National Institutes of Health | And 10 more authors.
Journal of Neurosurgery | Year: 2012

Object. Tumor-initiating cells are uniquely resilient to current treatment modalities and play an important role in tumor resistance and recurrence. The lack of specific tumor-initiating cell markers to identify and target these cells presents a major obstacle to effective directed therapy. Methods. To identify tumor-initiating cell markers in primary brain tumors, the authors compared the proteomes of glioma tumor-initiating cells to their differentiated progeny using a novel, nongel/shotgun-based, multidimensional liquid-chromatography protein separation technique. An in vivo xenograft model was used to demonstrate the tumorigenic and stem cell properties of these cells. Western blot and immunofluorescence analyses were used to confirm findings of upregulated ciliary neurotrophic factor receptor subunit-α (CNTFRα) in undifferentiated tumor-initiating cells and gliomas of increasing tumor grade. Sequencing of the CNTFRα coding regions was performed for mutation analysis. Finally, antibody-dependent cell-mediated cytotoxicity was used to establish the role of CNTFRα as a potential immunotherapeutic target. Results. Ciliary neurotrophic factor receptor subunit-α expression was increased in tumor-initiating cells and was decreased in the cells' differentiated progeny, and expression levels increased with glioma grade. Mutations of CNTFRα are not common in gliomas. Functional studies using CNTF treatment in glioma tumor-initiating cells showed induction of differentiation through the CNTFRα pathway. Treatment with anti-CNTFRα antibody resulted in increased antibody-dependent cell-mediated cytotoxicity in CNTFRα expressing DAOY cells but not in cell lines that lack CNTFRα. Conclusions. These data indicate that CNTFRa plays a role in the formation or maintenance of tumor-initiating cells in gliomas, is a marker that correlates with histological grade, may underlie treatment resistance in some cases, and is a potential therapeutic target.


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 | Wang C.,University of Maryland University College | Lee C.S.,University of Maryland University College
Methods in Molecular Biology | 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. © 2013 Springer Science+Business Media, LLC.


Fang X.,University of Maryland University College | Wang C.,University of Maryland University College | Balgley B.M.,Calibrant Biosystems | Zhao K.,University of Maryland University College | And 4 more authors.
Journal of Proteome Research | Year: 2012

Complicating proteomic analysis of whole tissues is the obvious problem of cell heterogeneity in tissues, which often results in misleading or confusing molecular findings. Thus, the coupling of tissue microdissection for tumor cell enrichment with capillary isotachophoresis-based selective analyte concentration not only serves as a synergistic strategy to characterize low abundance proteins, but it can also be employed to conduct comparative proteomic studies of human astrocytomas. A set of fresh frozen brain biopsies were selectively microdissected to provide an enriched, high quality, and reproducible sample of tumor cells. Despite sharing many common proteins, there are significant differences in the protein expression level among different grades of astrocytomas. A large number of proteins, such as plasma membrane proteins EGFR and Erbb2, are up-regulated in glioblastoma. Besides facilitating the prioritization of follow-on biomarker selection and validation, comparative proteomics involving measurements in changes of pathways are expected to reveal the molecular relationships among different pathological grades of gliomas and potential molecular mechanisms that drive gliomagenesis. © 2012 American Chemical Society.


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.


Xu D.S.,U.S. National Institutes of Health | Yang C.,U.S. National Institutes of Health | Proescholdt M.,University of Regensburg | Brundl E.,University of Regensburg | And 6 more authors.
PLoS ONE | Year: 2012

Glioblastoma multiforme is the most common and malignant primary brain tumor. Recent evidence indicates that a subset of glioblastoma tumor cells have a stem cell like phenotype that underlies chemotherapy resistance and tumor recurrence. We utilized a new "multidimensional" capillary isoelectric focusing nano-reversed-phase liquid chromatography platform with tandem mass spectrometry to compare the proteomes of isolated glioblastoma tumor stem cell and differentiated tumor cell populations. This proteomic analysis yielded new candidate proteins that were differentially expressed. Specifically, two isoforms of the membrane proteolipid neuronatin (NNAT) were expressed exclusively within the tumor stem cells. We surveyed the expression of NNAT across 10 WHO grade II and III gliomas and 23 glioblastoma (grade IV) human tumor samples and found NNAT was expressed in a subset of primary glioblastoma tumors. Through additional in vitro studies utilizing the U87 glioma cell line, we found that expression of NNAT is associated with significant increases in cellular proliferation. Paralleling the in vitro results, when NNAT levels were evaluated in tumor specimens from a consecutive cohort of 59 glioblastoma patients, the presence of increased levels of NNAT were found to be a an independent risk factor (P = 0.006) for decreased patient survival through Kaplan-Meier and multivariate analysis. These findings indicate that NNAT may have utility as a prognostic biomarker, as well as a cell-surface target for chemotherapeutic agents. © 2012 This is an open-access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.


PubMed | Calibrant Biosystems
Type: | Journal: Methods in molecular biology (Clifton, N.J.) | Year: 2012

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.

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