分类： 生物学 >> 生物物理学 提交时间： 2016-05-12
Cai, Tanxi
Shu, Qingbo
Liu, Peibin
Niu, Lili
Guo, Xiaojing
Yang, Fuquan
Cai, Tanxi
Shu, Qingbo
Liu, Peibin
Niu, Lili
Guo, Xiaojing
Yang, Fuquan
Cai, Tanxi
Shu, Qingbo
Hou, JunJie
Liu, Charles C.
摘要： Phosphoinositides, the phosphorylated derivatives of phosphatidylinositol (PtdIns), are key regulators of many fundamental biological processes, including cell growth, proliferation, and motility. Here, we present a novel method for rapid, sensitive, and simultaneous profiling of phosphatidylinositol trisphosphate (PtdInsP(3)), phosphatidylinositol bisphosphate (PtdInsP(2)), and phosphatidylinositol phosphate (PtdInsP) of different fatty acid compositions. This method is based on a technique called charged diacylglycerol fragment ion-specific multiple precursor ion scanning (DAG(+)-specific MPIS), coupled with prior phosphate methylation. Using DAG(+)-specific MPIS, we were able to identify 32 PtdIns, 28 PtdInsP, 30 PtdInsP(2), and 3 PtdInsP(3) molecular species from bovine brain extracts or prostatic cancer cell lines in an efficient and time-saving manner. Our analysis revealed a large range of fatty acyl compositions in phosphoinositides not obtained previously from mammalian samples. We also developed a method that involves isotopic labeling of endogenous phosphoinositides with deuterated diazomethane (CD2N2) for quantitation of phosphoinositides. CD2N2 was generated in situ through acid-catalyzed H/D exchange and methanolysis of trimethylsilyl diazomethane (TMS-diazomethane). Phosphoinositides, extracted from a PC3 prostatic cancer cell line, were labeled either with CH2N2 or CD2N2 and mixed in known proportions for DAG(+)-specific MPIS-based mass spectrometry (MS) analysis. The results indicate that isotopic labeling is capable of providing accurate quantitation of PtdInsP(3), PtdInsP(2), and PtdInsP with adequate linearity as well as high reproducibility with an average coefficient variation of 18.9%. More importantly, this new methods excluded the need for multiple phosphoinositide internal standards. DAG(+)-specific MPIS and isotopic labeling based MS analysis of phosphoinositides offers unique advantages over existing approaches and presents a powerful tool for research of phosphoinositide metabolism.
分类： 生物学 >> 生物物理学 提交时间： 2016-05-12
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
摘要： 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.
分类： 生物学 >> 生物物理学 >> 肿瘤学 提交时间： 2016-05-05
Zhang, Junjie
Guo, Fei
Wang, Lei
Zhao, Wei
Zhang, Da
Yang, Heying
Wang, Jiaxiang
Hu, Qian
Yu, Jiekai
Zheng, Shu
Niu, Lili
Yang, Fuquan
摘要： Wilms' tumors are one of the most common malignant, solid intra-abdominal tumors observed in children. Although potential tumor markers have been found, inflammatory cytokines interfere with the process of specific protein identification. The present study was undertaken to identify post-traumatic stress-related factors of Wilms' tumors and to verify the accuracy of early-stage tumor-specific serum protein markers. Serum samples were screened for differentially-expressed proteins using surface-enhanced laser desorption/ionization-time of flight mass spectrometry (SELDI-TOF-MS). Potential markers were isolated and purified using solid-phase extraction (SPE) and SDS-PAGE. Following enzymatic digestion of the protein samples, the peptide fragments were detected with high performance liquid chromatography-mass spectrometry. The obtained peptide mass fingerprint was searched in the Swiss-Prot protein sequence database via the Mascot search engine. Differentially-expressed proteins were verified using western blot analysis. Differentially-expressed proteins with a mass/charge of 5,816 were screened out using SELDI-TOF-MS, and significant differences between the tumor and control groups, and the trauma and control groups were observed. Target proteins were isolated and purified using SPE and SDS-PAGE. Thioredoxin 1 (Trx1) was found to be differentially expressed. In the serum of children with Wilms' tumors, there was an increase in the level of the post-traumatic stress-related inflammatory factor, Trx1, as compared with the normal control group. Thus, the results of this study indicate that Trx1 presents a potential post-traumatic stress-related factor of Wilms' tumors.
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