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傅里叶变换离子回旋共振质谱法分析雄激素抑制的人前列腺癌细胞磷酸化蛋白质组。

Characterization of the phosphoproteome in androgen-repressed human prostate cancer cells by Fourier transform ion cyclotron resonance mass spectrometry.

机构信息

Department of Chemistry and Biochemistry, 95 Chieftain Way, Florida State University, Tallahassee, Florida 32306, United States.

出版信息

J Proteome Res. 2011 Sep 2;10(9):3920-8. doi: 10.1021/pr2000144. Epub 2011 Jul 26.

Abstract

Androgen-repressed human prostate cancer, ARCaP, grows and is highly metastatic to bone and soft tissues in castrated mice. The molecular mechanisms underlying the aberrant responses to androgen are not fully understood. Here, we apply state-of-the-art mass spectrometry methods to investigate the phosphoproteome profiles in ARCaP cells. Because protein biological phosphorylation is always substoichiometric and the ionization efficiency of phosphopeptides is low, selective enrichment of phosphorylated proteins/peptides is required for mass spectrometric analysis of phosphorylation from complex biological samples. Therefore, we compare the sensitivity, efficiency, and specificity for three established enrichment strategies: calcium phosphate precipitation (CPP), immobilized metal ion affinity chromatography (IMAC), and TiO(2)-modified metal oxide chromatography. Calcium phosphate precipitation coupled with the TiO(2) approach offers the best strategy to characterize phosphorylation in ARCaP cells. We analyzed phosphopeptides from ARCaP cells by LC-MS/MS with a hybrid LTQ/FT-ICR mass spectrometer. After database search and stringent filtering, we identified 385 phosphoproteins with an average peptide mass error of 0.32 ± 0.6 ppm. Key identified oncogenic pathways include the mammalian target of rapamycin (mTOR) pathway and the E2F signaling pathway. Androgen-induced proliferation inhibitor (APRIN) was detected in its phosphorylated form, implicating a molecular mechanism underlying the ARCaP phenotype.

摘要

雄激素抑制的人前列腺癌(ARCaP)在去势小鼠中生长并高度转移至骨骼和软组织。雄激素异常反应的分子机制尚未完全阐明。在此,我们应用最先进的质谱方法研究 ARCaP 细胞的磷酸化蛋白质组谱。由于蛋白质生物磷酸化总是亚化学计量的,并且磷酸肽的离子化效率低,因此需要对来自复杂生物样品的磷酸化进行质谱分析时,需要选择性地富集磷酸化蛋白/肽。因此,我们比较了三种已建立的富集策略(磷酸钙沉淀(CPP)、固定化金属离子亲和色谱(IMAC)和 TiO2 修饰的金属氧化物色谱)的灵敏度、效率和特异性。磷酸钙沉淀与 TiO2 方法相结合,为表征 ARCaP 细胞中的磷酸化提供了最佳策略。我们使用 LTQ/FT-ICR 质谱仪通过 LC-MS/MS 分析 ARCaP 细胞中的磷酸肽。在数据库搜索和严格过滤后,我们鉴定了 385 种磷酸化蛋白,平均肽质量误差为 0.32±0.6 ppm。关键鉴定的致癌途径包括哺乳动物雷帕霉素靶蛋白(mTOR)途径和 E2F 信号通路。雄激素诱导增殖抑制剂(APRIN)以磷酸化形式被检测到,暗示了 ARCaP 表型的分子机制。

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