Publications (2)10.23 Total impact
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Article: Comprehensive phosphoproteome analysis of INS-1 pancreatic beta-cells using various digestion strategies coupled with liquid chromatography-tandem mass spectrometry.
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ABSTRACT: Type 2 diabetes results from aberrant regulation of the phosphorylation cascade in beta-cells. Phosphorylation in pancreatic beta-cells has not been examined extensively, except with regard to subcellular phosphoproteomes using mitochondria. Thus, robust, comprehensive analytical strategies are needed to characterize the many phosphorylated proteins that exist, because of their low abundance, the low stoichiometry of phosphorylation, and the dynamic regulation of phosphoproteins. In this study, we attempted to generate data on a large-scale phosphoproteome from the INS-1 rat pancreatic beta-cell line using linear ion trap MS/MS. To profile the phosphoproteome in-depth, we used comprehensive phosphoproteomic strategies, including detergent-based protein extraction (SDS and SDC), differential sample preparation (in-gel, in-solution digestion, and FASP), TiO2 enrichment, and MS replicate analyses (MS2-only and multiple-stage activation). All spectra were processed and validated by stringent multiple filtering using target and decoy databases. We identified 2467 distinct phosphorylation sites on 1419 phosphoproteins using 4 mg of INS-1 cell lysate in 24 LC-MS/MS runs, of which 683 (27.7%) were considered novel phosphorylation sites that have not been characterized in human, mouse, or rat homologues. Our informatics data constitute a rich bioinformatics resource for investigating the function of reversible phosphorylation in pancreatic beta-cells. In particular, novel phosphorylation sites on proteins that mediate the pathology of type 2 diabetes, such as Pdx-1, Nkx.2, and Srebf1, will be valuable targets in ongoing phosphoproteomics studies.Journal of Proteome Research 01/2012; 11(4):2206-23. · 5.11 Impact Factor -
Article: Detection of differential proteomes associated with the development of type 2 diabetes in the Zucker rat model using the iTRAQ technique.
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ABSTRACT: Type 2 diabetes (T2D) is closely associated with obesity, and it arises when pancreatic β cells fail to achieve β cell compensation. However, the mechanism linking obesity, insulin resistance, and β cell failure in T2D is not fully understood. To explore this association, we carried out a differential proteomics study using the disease models of Zucker Fatty (ZF) and Zucker Diabetic Fatty (ZDF) rats as the rat models for obese/prediabetes and obese/diabetes, respectively. Differentially expressed islet proteins were identified among ZDF, ZF, and Zucker Lean (ZL, control rat) rats using three iTRAQ experiments, where three biological replicates and two technical replicates were examined to assess both the technical and biological reproducibilities. A total of 54 and 58 proteins were differentially expressed in ZDF versus ZL rats and in ZF versus ZL rats, respectively. Notably, the novel proteins involved in impaired insulin secretion (Scg2, Anxa2, and Rab10), mitochondrial dysfunction (Atp5b and Atp5l), extracellular matrix proteins (Lgal-1, Vim, and Fbn1), and microvascular ischemia (CPA1, CPA2, CPB, Cela2a, and Cela3b) were observed for the first time. With these novel proteins, our proteomics study could provide valuable clues for better understanding the underlying mechanisms associated with the dynamic transition of obesity to T2D.Journal of Proteome Research 02/2011; 10(2):564-77. · 5.11 Impact Factor
