Mass Spectrometry Methods for Probing Metabolic Dynamics in Normal & Cancer Cells

描述(申请人提供)：许多临床上最重要的化疗药物抑制肿瘤细胞的新陈代谢。我们的首要目标是全面和定量地了解正常细胞和癌细胞之间的代谢差异，并利用这些知识来指导新的抗癌方案的合理设计。为了实现这一目标，我们正在开发应用最先进的液相色谱-电喷雾电离-三重四极杆质谱仪技术来动态、定量和全面地探测细胞代谢的方法。到目前为止，我们已经成功地开发了测量几种微生物的代谢物浓度和通量的方法。在这里，我们建议扩展这些方法，以便能够可靠地测量正常细胞和癌细胞中代谢物的浓度和通量。具体地说，我们的目标是能够量化至少150种不同的已知的、结构上定义的细胞内代谢物的浓度。我们还打算使用同位素示踪剂，测量通过中心碳、脂、氨基酸和核苷酸代谢的通量。我们将应用我们开发的分析技术来绘制正常细胞和癌细胞之间的主要代谢差异，并研究这些细胞对抗代谢抗癌药物治疗的动态反应。这些方法将对了解抗代谢抗癌药物的作用机制(和毒性)，表征药物敏感和耐药癌细胞之间的代谢差异，以及提出抑制新陈代谢的新方法，从而特异性地杀死癌细胞具有长期价值。

DESCRIPTION (provided by applicant): Many of the most clinically important chemotherapeutic agents inhibit the metabolism of tumor cells. Our overarching goal is to develop a complete and quantitative understanding of the metabolic differences between normal and cancer cells, and to use this knowledge to guide the rational design of novel anticancer regimens. To achieve this goal, we are developing methods that apply state-of-the-art liquid chromatography- electrospray ionization-triple quadrupole mass spectrometry technology to probe cellular metabolism in a dynamic, quantitative, and comprehensive manner. To date, we have succeeded in developing methods for measuring metabolite concentrations and fluxes from several microbes. Here we propose to extend these methods to enable reliable measurement of metabolite concentrations and fluxes in normal and cancer cells. Specifically, we aim to enable quantitation of the concentrations of at least 150 different known, structurally-defined intracellular metabolites. We also aim to measure, using isotopic tracers, the fluxes through central carbon, lipid, amino acid, and nucleotide metabolism. We will apply the analytical technology that we develop to map major metabolic differences between normal and cancer cells and to study the dynamic response of these cells to treatment with anti-metabolite anticancer drugs. The methods developed here will have long-term value for understanding the mechanism of action (and toxicity) of anti-metabolite anticancer drugs, for characterizing the metabolic differences between drug-responsive and resistant cancer cells, and for suggesting new approaches to inhibiting metabolism that will specifically kill cancer cells.