Indianapolis, IN, United States
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Kim J.W.,Indiana University | Nie B.,Indiana University | Sahm H.,Indiana University | Brown D.P.G.,Indiana University | And 4 more authors.
Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences | Year: 2010

Protein quantification in a complex protein mixture presents a daunting task in biochemical analysis. Antibody-based immunoassays are traditional methods for protein quantification. However, there are issues associated with accuracy and specificity in these assays, especially when the changes are small (e.g., <2-fold). With recent developments in mass spectrometry, monitoring a selected peptide, thus protein, in a complex biological sample has become possible. In this study, we demonstrate a simple mass spectrometry-based method for selective measurement of a moderately low abundant protein, superoxide dismutase 1 (SOD1), in cisplatin-sensitive and cisplatin-resistant human ovarian cancer cells. Selected-reaction-monitoring (SRM) technology was employed to specifically analyze the target peptides in a pair of human ovarian cancer cell lines: 2008/2008-C13*5.25 (cisplatin-sensitive/cisplatin-resistant, respectively). The observed 1.47-fold higher expression in the resistant cell line is consistent with findings by other approaches. This robust liquid chromatography/mass spectrometry (LC/MS) method provides a powerful tool for targeted proteomic verification and/or validation studies. © 2010 Elsevier B.V. All rights reserved.

Kim J.W.,Indiana University | Sahm H.,Indiana University | You J.,Monarch LifeSciences LLC | Wang M.,Indiana University | Wang M.,Monarch LifeSciences LLC
Anticancer Research | Year: 2010

Background: Overexpression of superoxide dismutase 1 (SOD1) has been shown to be one of the factors involved in causing cisplatin resistance in ovarian cancer. Reduction of SOD1 expression is expected to restore, at least partially, cisplatin sensitivity in ovarian cancer chemotherapy. Here, we explored the potential of RNAi as a therapy for reversal of cisplatin resistance. Materials and Methods: SOD1-specific small-interfering RNA (siRNA) was synthesized and transfected into cisplatin-resistant cell line A2780/CP prior to treatment with 15 μM cisplatin. Cell survival was assessed by clonogenic assay. Results: An enhanced cisplatin sensitivity was observed in the A2780/CP cells treated with SOD1-specific siRNA, compared to non-siRNA-treated or scrambled-siRNA-treated control cells. Conclusion: Specifically targeting SOD1 could lead to sensitization of cisplatin-resistant ovarian cancer cells, and SOD1 may be used as a potential target for chemosensitizers.

Doyle C.J.,Indiana University | Yancey K.,Indiana University | Pitt H.A.,Indiana University | Wang M.,Indiana University | And 6 more authors.
Pancreas | Year: 2012

OBJECTIVES: The aims of this study were to characterize the proteome of normal pancreatic juice, to analyze the effect of secretin on the normal proteome, and to compare these results with published data from patients with pancreatic cancer. METHODS: Paired pancreatic fluid specimens (before and after intravenous secretin stimulation) were obtained during endoscopic pancreatography from 3 patients without significant pancreatic pathology. Proteins were identified and quantified by mass spectrometry-based protein quantification technology. The human RefSeq (NCBI) database was used to compare the data in samples from patients without pancreatic disease with published data from 3 patients with pancreatic cancer. RESULTS: A total of 285 proteins were identified in normal pancreatic juice. Ninety had sufficient amino acid sequences identified to characterize the protein with a high level of confidence. All 90 proteins were present before and after secretin administration but with altered relative concentrations, usually by 1 to 2 folds, after stimulation. Comparison with 170 published pancreatic cancer proteins yielded an overlap of only 42 proteins. CONCLUSIONS: Normal pancreatic juice contains multiple proteins related to many biological processes. Secretin alters the concentration but not the spectrum of these proteins. The pancreatic juice proteome of patients without pancreatic disease and that of patients with pancreatic cancer differ markedly. Copyright © 2012 by Lippincott Williams & Wilkins.

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