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1. chinaXiv:201605.01438 [pdf]

Quantitative proteomics using SILAC: Principles, applications, and developments

Chen, Xiulan; Wei, Shasha; Ji, Yanlong; Guo, Xiaojing; Yang, Fuquan; Chen, Xiulan; Wei, Shasha; Ji, Yanlong; Guo, Xiaojing; Yang, Fuquan; Ji, Yanlong
Subjects: Biology >> Biophysics

SILAC is based on direct addition of selected stable isotope amino acids into the cell culture medium, allowing superior quantitative analysis of the cellular proteome compared to other labeling methods. The great advantages of SILAC lie in its straight-forward implementation, quantitative accuracy, and reproducibility over chemical labeling or label-free quantification strategies, favoring its adoption for proteomic research. SILAC has been widely applied to characterize the proteomic changes between different biological samples, to investigate dynamic changes of protein PTMs, to distinguish specific interacting proteins in interaction proteomic analysis, and to analyze protein turnover in the proteome-wide scale. The present review summarizes the principles of SILAC technology, its applications in biological research, and the present state of this technology.

submitted time 2016-05-12 Hits534Downloads362 Comment 0

2. chinaXiv:201605.01392 [pdf]

Proteomic Comparison and MRM-Based Comparative Analysis of Metabolites Reveal Metabolic Shift in Human Prostate Cancer Cell Lines

Shu, Qingbo; Cai, Tanxi; Chen, Xiulan; Xue, Peng; Zhu, Nali; Xie, Zhensheng; Wei, Shasha; Niu, Lili; Yang, Fuquan; Shu, Qingbo; Cai, Tanxi; Chen, Xiulan; Xue, Peng; Zhu, Nali; Xie, Zhensheng; Wei, Shasha; Niu, Lili; Yang, Fuquan; Shu, Qingbo; Zhang, Qing
Subjects: Biology >> Biophysics

One of the major challenges in prostate cancer therapy remains the development of effective treatments for castration-resistant prostate cancer (CRPC), as the underlying mechanisms for its progression remain elusive. Previous studies showed that androgen receptor (AR) is crucially involved in regulation of metabolism in prostate cancer (PCa) cells throughout the transition from early stage, androgen-sensitive PCa to androgen-independent CRPC. AR achieves such metabolic rewiring directively either via its transcriptional activity or via interactions with AMP-activated protein kinase (AMPK). However, due to the heterogeneous expression and activity status of AR in PCa cells, it remains a challenge to investigate the links between AR status and metabolic alterations. To this end, we compared the proteomes of three pairs of androgen-sensitive (AS) and androgen-independent (AI) PCa cell lines, namely, PC3-AR(+)/PC3, 22Rv1/Du145, and LNCaP/C42B, using an iTRAQ labeling approach. Our results revealed that most of the differentially expressed proteins between each pair function in metabolism, indicating a metabolic shift between AS and AT cells, as further validated by multiple reaction monitoring (MRM)-based quantification of nucleotides and relative comparison of fatty acids between these cell lines. Furthermore, increased adenylate kinase isoenzyme 1 (AK1) in AS relative to AT cells may result in activation of AMPK, representing a major regulatory factor involved in the observed metabolic shift in PCa cells.

submitted time 2016-05-12 Hits467Downloads282 Comment 0

3. chinaXiv:201605.00707 [pdf]

Comparative Proteomic Study of Fatty Acid-treated Myoblasts Reveals Role of Cox-2 in Palmitate-induced Insulin Resistance

Chen, Xiulan; Wei, Shasha; Yang, Fuquan; Chen, Xiulan; Wei, Shasha; Yang, Fuquan; Xu, Shimeng; Deng, Yaqin; Li, Yiran; Liu, Pingsheng; Xu, Shimeng; Wei, Shasha; Deng, Yaqin; Li, Yiran;
Subjects: Biology >> Biophysics

Accumulated studies demonstrate that saturated fatty acids (FAs) such as palmitic acid (PA) inhibit insulin signaling in skeletal muscle cells and monounsaturated fatty acids such as oleic acid (OA) reverse the effect of PA on insulin signaling. The detailed molecular mechanism of these opposite effects remains elusive. Here we provide a comparative proteomic study of skeletal myoblast cell line C2C12 that were untreated or treated with PA, and PA plus OA. A total of 3437 proteins were quantified using SILAC in this study and 29 proteins fall into the pattern that OA reverses PA effect. Expression of some these proteins were verified using qRT-PCR and Western blot. The most significant change was cyclooxygenase-2 (Cox-2). In addition to whole cell comparative proteomic study, we also compared lipid droplet (LD)-associated proteins and identified that Cox-2 was one of three major altered proteins under the FA treatment. This finding was then confirmed using immunofluorescence. Finally, Cox-2 selective inhibitor, celecoxib protected cells from PA-reduced insulin signaling Akt phosphorylation. Together, these results not only provide a dataset of protein expression change in FA treatment but also suggest that Cox-2 and lipid droplets (LDs) are potential players in PA- and OA-mediated cellular processes.

submitted time 2016-05-05 Hits301Downloads197 Comment 0

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