• 主题
高级搜索  >
历史搜索 清空历史
首页> 外文学位 >Absolute quantitation of intracellular metabolites and metabolic fluxes via liquid chromatography-mass spectrometry.
【24h】

Absolute quantitation of intracellular metabolites and metabolic fluxes via liquid chromatography-mass spectrometry.

机译：通过液相色谱-质谱法对细胞内代谢产物和代谢通量进行绝对定量。

获取原文
获取原文并翻译 | 示例
页面导航
  • 摘要
  • 著录项
  • 相似文献
  • 相关主题

摘要

Methods were developed for the measurement of absolute metabolite concentrations, as well as metabolic fluxes in cultured cells. Absolute metabolite concentrations were determined using isotopically labeled cell extracts and unlabeled standards. Flux determination was performed by monitoring the dynamic incorporation of stable-isotope labeled compounds along with knowledge of the metabolite concentrations. Both approaches used the measurement of isotopomer ratios, monitored by a liquid chromatograph tandem mass spectrometry approach. The concentrations of over one hundred intracellular metabolites in the model organism Escherichia coli were determined. These concentrations were used to evaluate the expected thermodynamics and kinetics of reaction based upon the previously known free energy of reactions and binding constants of enzymes, finding that the majority of enzyme active sites were saturated for substrate during exponential growth.;During Human Cytomegalovirus (HCMV) infection, human fibroblasts significantly alter their metabolism. In order to quantify these changes, approximately 30 intracellular concentrations were determined, and a battery of experiments monitoring the rate of isotope incorporation were performed. By fitting a series of differential equations to these data, quantitative estimates of cellular metabolic fluxes in both HCMV infected and uninfected cells were determined. Metabolic flux into fatty acid biosynthesis was found to be significantly increased during infection, and inhibition of this pathway by small molecules significantly reduced the ability of HCMV to reproduce.
机译：开发了用于测量绝对代谢物浓度以及培养细胞中代谢通量的方法。使用同位素标记的细胞提取物和未标记的标准液确定绝对代谢物浓度。通过监测稳定同位素标记的化合物的动态掺入以及代谢物浓度的知识来进行通量测定。两种方法都使用了同位素异构体比率的测量方法，该方法通过液相色谱串联质谱法进行监测。测定了模型生物大肠杆菌中一百多种细胞内代谢物的浓度。根据先前已知的反应自由能和酶的结合常数，将这些浓度用于评估预期的反应热力学和动力学，发现在指数生长期间，大部分酶活性位点对于底物而言是饱和的。 ）感染后，人成纤维细胞会明显改变其代谢。为了量化这些变化，确定了大约30种细胞内浓度，并进行了一系列监测同位素掺入速率的实验。通过将一系列微分方程拟合到这些数据，可以确定HCMV感染和未感染细胞中细胞代谢通量的定量估计。发现在感染过程中进入脂肪酸生物合成的代谢通量显着增加，并且小分子对该途径的抑制显着降低了HCMV繁殖的能力。

著录项

  • 作者

    Bennett, Bryson Donald.;

  • 作者单位

    Princeton University.;

  • 授予单位 Princeton University.;
  • 学科 Chemistry Biochemistry.;Biology Virology.
  • 学位 Ph.D.
  • 年度 2010
  • 页码 253 p.
  • 总页数 253
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类
  • 关键词

相似文献

  • 外文文献
  • 中文文献
  • 专利
获取原文

客服邮箱：kefu@zhangqiaokeyan.com

京公网安备：11010802029741号 ICP备案号：京ICP备15016152号-6 六维联合信息科技 (北京) 有限公司©版权所有

  • 客服微信

  • 服务号