Resource for the Development of Biomedical Accelerator Mass Spectrometry (AMS)

描述（由申请人提供）：自1999年成立以来，NIH资助的加速器质谱（AMS）生物医学应用研究资源在许多领域进行了许多突破性研究，包括生物分子周转，内源性化合物和外源性物质的定量代谢以及潜在致突变加合物的分析。AMS的高灵敏度、准确度和精密度使这些研究成为可能。在这个TR&D核心中提出的工作将建立在成功的高影响力研究的基础上，并将扩展当前AMS实验和分析方法的适用性，以应对更广泛的科学和技术挑战。该核心的重点是蛋白质和其他生物大分子绝对定量方法的开发和常规实施。该核心将支持几个驱动生物医学项目，包括：1）表征由于人为大气14 C释放而标记的组织和细胞类型的周转率（炸弹脉冲生物学）; 2）蛋白质翻译后修饰的绝对定量; 3）基于14 C-配体结合的受体鉴定;和4）开发标记策略、分离技术和分析程序，用于定量生物基质中的计算设计的合成蛋白质药物，以支持动物给药实验和人类微量给药研究。 生物大分子在细胞、组织和生物体中发挥着广泛的重要作用。蛋白质是所有生物体的基本组成部分，蛋白质酶催化生命的化学反应。核酸提供遗传物质，其中包含构建、维持和调节生物体的信息。由于生物大分子发挥着如此重要和多样的作用，我们定量追踪大分子及其修饰的能力的提高将使重要的发现成为可能，这将大大促进我们对生物过程的理解。中 最近的论文，Hanke等人（Hanke等人，2008）写道： “最终，非常希望获得系统中每种蛋白质的精确定量值，例如，它们的拷贝数/细胞或它们的浓度（纳克/毫升体液）。虽然这种关于蛋白质的基本信息本身对生物学家来说已经很有价值，但系统生物学甚至需要它作为建模的输入。在医学背景下，了解血液中某些蛋白质或其他常见生物标志物来源的确切数量可以为患者治疗提供诊断相关信息。" 蛋白质和其他生物大分子的分析测量提出了许多重大的技术挑战，特别是当需要定量时。从生物基质中提取大分子通常比提取小分子更复杂，并且提取物更易于污染和降解。大分子如蛋白质和核酸是异质分子，并且可以由于酶介导的反应而被化学修饰（例如，翻译后 修饰）或作为组织处理的结果，如在福尔马林固定的组织的情况下。此外，生物大分子可能易于通过物理或化学手段裂解。虽然分析标准品通常可以购买或合成用于感兴趣的小分子，但生物大分子很少有类似的标准品。测量生物大分子的绝对量的能力与生物学和医学具有广泛的相关性，因为目前还没有标准化的通用分析方法能够进行这些测量。该核心的主要重点是开发应用AMS的方法 该技术，特别是与新的液体样品AMS接口相结合，克服了生物大分子分析的这些主要挑战。为此，我们建议与合作研究人员互动，以解决各种重要的生物学问题。

DESCRIPTION (provided by applicant): Since its inception in 1999, the NIH-funded Research Resource on the biomedical applications of accelerator mass spectrometry (AMS) has performed numerous groundbreaking studies in many areas including biomolecule turnover, quantitative metabolism of endogenous compounds and xenobiotics, and analysis of potentially mutagenic adducts. These studies were made possible by the high sensitivity, accuracy, and precision of AMS. The work proposed in this TR&D core will build on this legacy of successful, high impact research and will extend the applicability of current AMS experimental and analytical methods to a wider set of scientific and technical challenges. The focus for this core i the development and routine implementation of methods for absolute quantitation of proteins and other biological macromolecules. This core will support several driving biomedical projects, including: 1) Characterization of turnover rates of tissues and cell types labeled as a result of anthropogenic atmospheric 14C release (bomb-pulse biology); 2) Absolute quantitation of protein post-translational modifications; 3) Identification of receptors based on 14C-ligand binding; and 4) Development of labeling strategies, separation techniques, and analytical procedures for quantitation of computationally-designed synthetic protein drugs in biological matrices, in support of animal dosing experiments and human microdosing studies. Biological macromolecules play a wide variety of essential roles in cells, tissues, and organisms. Proteins are the basic building blocks of all living organisms, and proteinaceous enzymes catalyze the chemical reactions responsible for life. Nucleic acids provide the genetic material containing information for building, maintaining, and regulating living organisms. Because biological macromolecules play such essential and diverse roles, improvements in our ability to quantitatively trace macromolecules and their modifications will enable important discoveries that will significantly advance our understanding of biological processes. In a recent paper, Hanke et al (Hanke et al., 2008) wrote: "Ultimately, it would be highly desirable to obtain exact quantitative values of each protein in a system, e.g., their copy number per cell or their concentration in nanogram per milliliter of body fluid. While this kind of basic information about a protein is already per se valuable for the biologist, systems biology even requires it as input for modeling. In a medical context, knowing the exact amounts of certain proteins in blood or other common sources of biomarkers can provide diagnostically relevant information for patient treatment." Analytical measurement of proteins and other biological macromolecules presents a number of significant technical challenges, particularly when quantitation is required. Macromolecule extraction from biological matrices is typically more complex than the extraction of small molecules, and extracts are more prone to contamination and degradation. Macromolecules such as proteins and nucleic acids are heterogeneous molecules, and may be chemically modified as a result of enzyme-mediated reactions (e.g., post-translational modifications) or as a result of tissue processing, as in the case of formalin-fixed tissue. In addition, biological macromolecules may be susceptible to cleavage by physical or chemical means. While analytical standards can usually be purchased or synthesized for small molecules of interest, similar standards are rarely available for biological macromolecules. The ability to measure absolute quantities of biological macromolecules has broad relevance for biology and medicine, as there currently is no standardized, universal analytical method capable of making these measurements. The major focus of this core is to develop methods for applying AMS technology, especially in conjunction with the new liquid sample AMS interface, to overcome these major challenges of biological macromolecule analysis. To this end, we propose to interact with collaborating researchers to solve a variety of important biological problems.