Instrumentation Development: MS Array for Quantitative Proteomics

描述(由申请人提供)：基因组测序技术的快速进步极大地促进了在分子水平上研究人类疾病。然而，有效利用当前和未来基因组信息的主要障碍与基因功能分配所固有的主要挑战有关。蛋白质组学领域的出现是为了帮助解决这一需求，因为蛋白质是基因表达的主要功能产物。蛋白质组含量的测量，包括蛋白质表达水平、翻译后修饰和蛋白质相互作用，可以产生与基因功能相关的关键见解。蛋白质组学研究中的主要挑战涉及样品的复杂性和必须进行测量的动态范围。一般而言，这些需求远远大于在基因组学中遇到的，因为蛋白质丰度的变化比基因大得多，蛋白质在物理性质上的多样性比基因大得多，蛋白质没有信号放大的手段，也没有与基因类似的沃森-克里克碱基配对。因此，蛋白质组学领域使用的技术主要基于多肽的质谱学测量，因为这些测量已经显示出大规模蛋白质识别和定量的能力。然而，由于每种蛋白质平均可以产生50-100个多肽，因此对多肽混合物的测量比对蛋白质的测量复杂得多。此外，目前依赖数据的测量策略导致可实现的动态范围显著压缩，因为这种MS/MS测量通常只适用于观察到的丰度较高的多肽。理想情况下，蛋白质组范围的消化中的每一个多肽都将受到MS/MS的影响，以从蛋白质组学实验中获得最大的信息。该项目将通过开发能够获取MS/MS的质谱计阵列来提高大规模蛋白质组学的能力，比目前最先进的技术快一个数量级。在该项目下开发的MS阵列技术将涉及离子回旋共振质谱仪，它将产生更高的质量分辨率、更高的质量测量精度以及更高的吞吐量采集。因此，在给定的实验过程中，可以识别一个或多个数量级的多肽，这将极大地增加每个蛋白质组分析的信息量，以及可以研究的蛋白质的动态范围。 与公共健康相关：蛋白质是疾病和正常健康状态下所有重要过程中的功能分子。该项目将开发新技术，通过在人体细胞、血清和其他生物液中识别至少一个数量级的蛋白质、翻译后修饰和蛋白质相互作用，从而提高对癌症、心血管疾病和神经疾病等人类疾病的理解。

DESCRIPTION (provided by applicant): The study of human diseases at the molecular level has benefitted greatly by rapid advances in technology for genome sequencing. However, major impediments for effective utilization of current and future genomic information relates to the major challenges that are inherent in functional assignment of genes. The field of proteomics has arisen to help address this need, since proteins are the predominant functional outcome of gene expression. Measurement of proteomic content, including protein expression levels, posttranslational modifications and protein interactions can yield critical insight relevant to gene function. The major challenges in proteomics research relate to the complexity of samples and the dynamic range over which measurements must be performed. In general terms, these demands are far greater than encountered in genomics since, protein abundances can vary much more than gene do, proteins have much wider diversity in physical properties than genes do, and proteins have no means for signal amplification, nor analogous Watson-Crick base pairing as genes do. Thus the field of proteomics employs technology largely based on mass spectrometry measurements of peptides since these measurements have shown capabilities for large-scale protein identification and quantitation. However, since each protein can produce on average, 50-100 peptides, measurements of peptide mixtures are far more complex than measurements on proteins. In addition, current data-dependent measurement strategies lead to significant compression of achievable dynamic range, since such MS/MS measurements are only normally feasible on peptides observed with higher abundance. Ideally, every peptide in a proteome-wide digest would be subjected to MS/MS to gain maximal information from proteomics experiments. This project will advance capabilities for large-scale proteomics through the development of a mass spectrometer array capable of MS/MS acquisition an order of magnitude faster than current state-of-the art technology. The MS array technology to be developed under this project will involve ion cyclotron resonance mass spectrometry that will yield higher mass resolving power, higher mass measurement accuracy as well as higher throughput acquisition. As a result, an order of magnitude or more peptides can be identified during a given experiment which will dramatically increase the information content of each proteomics analysis as well as the dynamic range of proteins that can be studied. PUBLIC HEALTH RELEVANCE: Proteins are the functional molecules in all important processes in disease and normal healthy state. This project will develop new technology that will enable improved understanding of human diseases such as cancer, cardiovascular disease, and neurological diseases by allowing identification of at least an order of magnitude more proteins, posttranslational modifications and protein interactions in human cells, serum and other biological fluids.