QUANTITATIVE METALLOPROTEOMICS: THE HUMAN ERYTHROCYTE

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内的代谢物和蛋白质可作为生物标志物来揭示许多疾病、代谢功能、营养状况和有毒金属暴露。虽然红细胞的蛋白质组学研究已经蓬勃发展，但红细胞的代谢组学研究仍处于起步阶段。在RBC正常功能所需的关键代谢物中，有金属/类金属，它们作为每一类细胞大分子的活化或结构辅因子。评估RBC金属含量是一项标准的临床测试，但令人惊讶的是，RBC中许多痕量金属和类金属的参考值并不为人所知。此外，许多被认为在功能上相关的金属/类金属没有鉴定出同源结合蛋白，因此它们的作用机制仍然不清楚。因此，建立RBC内所有金属/类金属（即金属组）的完整参考范围和所有金属/类金属结合蛋白（即金属蛋白组）的目录对于充分的临床实用性至关重要。到目前为止，技术障碍阻碍了实现这些目标的进展，但我们最近开发了将超灵敏元素分析与高通量蛋白质组学工具相结合的技术，从而能够分离和识别数百种蛋白质，并随后揭示结合的金属/类金属。本提案的总体目标是（目标1）定义人RBC金属组和（目标2）定义人RBC金属蛋白组。将以完整和亚细胞部分分析来自健康人供体的成熟RBC。蛋白质将通过等电点和分子量在液相中分离，然后通过MALDI-TOF分析和数据库匹配进行鉴定。通过ICP和STIM/PIXE的组合，将进行几乎涵盖整个周期表的元素分析，以达到RBC中万亿分之一的水平和分离蛋白质中百万分之一的水平。金属蛋白质组将通过合并蛋白质组和元素图谱来确定。结果将在一个供科学界检索的联机数据库中加以总结和公布。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。这项工作还将为更大规模的研究提供必要的基础，以确定疾病状态，次优营养和有毒金属暴露对RBC金属组和金属蛋白质组的影响，这可能会改善临床诊断并揭示新的治疗靶点。

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.