QUANTITATIVE METALLOPROTEOMICS: THE HUMAN ERYTHROCYTE

定量金属蛋白质组学：人类红细胞

• 批准号: 6957234
• 负责人: Bruce N Ames
• 金额: $ 23.67万
• 依托单位: CHILDREN'S HOSPITAL & RES CTR AT OAKLAND
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-09-01 至 2007-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6957234
• 关键词: Internet; biomarker; clinical research; erythrocytes; human tissue; information dissemination; isoelectric point; matrix assisted laser desorption ionization; metabolomics; metalloproteins; molecular biology information system; molecular weight; protein purification; protein quantitation /detection; proteomics

DESCRIPTION (provided by applicant): Red blood cells (RBCs) are a major diagnostic tool for important clinical data, as the metabolites and proteins within RBCs act as biomarkers to reveal numerous diseases, metabolic functions, nutritional status, and toxic metal exposure. While the proteomic study of the RBC has flourished, the metabolomic study of the RBC is still in its infancy. Among the critical metabolites required by RBC for proper function are the metals/metalloids that act as activating or structural cofactors for every class of cellular macromolecule Assessing RBC metal content is a standard clinical test, yet reference values for many of the trace metals and metalloids in the RBC are surprisingly not well known. Additionally, many metals/metalloids that are thought to be functionally relevant do not have identified cognate binding proteins, and thus their mechanisms; of action remain obscure. Therefore, establishing complete reference ranges for all metals/ metalloids (i.e. the metallome) and catalog of all metal/metalloid-binding proteins (i.e. the metalloproteome) within the RBC is essential for full clinical utility. Until now, technical obstacles prevented progress towards these goals, but we have recently developed technology that combines ultrasensitive elemental analysis with high-throughput proteomic tools, resulting in the capacity to separate and identify hundreds of proteins and to subsequently reveal bound metals/metalloids. The overall goal of this proposal is to (Aim 1) define the human RBC metallome and (Aim 2) define the human RBC metalloproteome. Mature RBCs from healthy human donors will be analyzed in whole and subcellular fractions. Proteins will be separated in liquid phase by isoelectric point and molecular weight and then identified by MALDI-TOF analysis and database matching. Elemental analysis spanning nearly the entire periodic table will be conducted to part per trillion levels in RBCs and part per million levels in separated proteins by combination of ICP and STIM/PIXE. The metalloproteome will be determined by merging proteomic and elemental maps. Results will be summarized and published in an on-line searchable database for the scientific community. This work will ultimately help integrate RBC proteomics and metabolomics for a better understanding of the role of RBCs in whole body metal homeostasis. This work will also provide the necessary foundation for larger studies to determine the effects of disease states, suboptimal nutrition, and toxic metal exposures on the RBC metallome and metalloproteome, which are likely to improve clinical diagnostics and reveal new therapeutic targets.

描述（由申请人提供）： 红细胞（RBC）是重要临床数据的主要诊断工具，因为RBC中的代谢产物和蛋白质充当生物标志物，可揭示许多疾病，代谢功能，营养状态和有毒金属暴露。尽管RBC的蛋白质组学研究蓬勃发展，但RBC的代谢组研究仍处于起步阶段。 RBC为正确功能所需的关键代谢产物中的金属/金属体物是对每类细胞大分子的激活或结构辅因子评估RBC金属含量的激活或结构辅因子，这是标准的临床测试，但对于RBC中许多痕量金属和金属体的参考值是令人惊讶的。此外，许多被认为与功能相关的金属/金属差尚未鉴定出结合蛋白，因此没有鉴定其机制。行动仍然晦涩难懂。因此，在RBC中建立所有金属/金属（即金属组）和所有金属/金属结合蛋白（即金属蛋白蛋白）的目录的完整参考范围对于完全临床效用至关重要。到目前为止，技术障碍阻止了这些目标的进步，但是我们最近开发了将超敏感性分析与高通量蛋白质组学工具相结合的技术，从而有能力分离和识别数百种蛋白质，并随后揭示了绑定的金属/金属素。该提案的总体目标是（目标1）定义人类RBC金属材料，并（AIM 2）定义人类RBC金属蛋白质组。全面和亚细胞分数将分析来自健康人类捐助者的成熟RBC。蛋白质将通过等电点和分子量在液相分离，然后通过MALDI-TOF分析和数据库匹配来鉴定。通过ICP和STIM/PIXE组合，将几乎整个元素周期表的元素分析将几乎整个元素周期表进行至每万亿级的一部分，而分离的蛋白质中的元素分析将在分离的蛋白质中进行。金属蛋白组将通过合并蛋白质组学和元素图来确定。结果将在科学界的在线可搜索数据库中进行总结和发布。这项工作最终将有助于整合RBC蛋白质组学和代谢组学，以更好地了解RBC在全身金属稳态中的作用。这项工作还将为大型研究提供必要的基础，以确定疾病状态，次优营养以及有毒金属暴露对RBC金属蛋白组和金属蛋白蛋白酶的影响，这些金属蛋白质可能会改善临床诊断并揭示新的治疗靶标。