Instrumentation Development: MS Array for Quantitative Proteomics

项目总结/摘要 在分子水平上对人类疾病的研究极大地受益于技术的快速进步， 基因组测序然而，有效利用当前和未来基因组信息的障碍 涉及与基因功能分配相关的挑战。蛋白质组学领域已经兴起， 因为蛋白质是细胞中主要的功能分子，测量 蛋白质组学内容，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重要见解。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宽动态范围。一般来说 这些需求远远大于基因组学中遇到的需求，因为蛋白质丰度可能差异很大 蛋白质的物理性质比基因的多样性要大得多， 没有信号放大的手段，也没有类似的沃森-克里克碱基配对的基因。因此， 的蛋白质组学采用的技术主要是基于质谱测量的肽，因为这些 测量已经显示出大规模蛋白质鉴定和定量的能力。但由于 每个蛋白质平均可以产生50-100个肽，肽混合物的测量要多得多。 比测量蛋白质复杂。此外，目前依赖数据的测量策略导致 可实现的动态范围的显著压缩，因为这样的MS/MS测量通常仅 对观察到的具有更高丰度的肽是可行的。理想情况下，蛋白质组范围内消化的每一种肽 进行MS/MS以从蛋白质组学实验中获得最大信息。该项目将推进 大规模蛋白质组学的能力，通过开发一个质谱仪阵列，能够高 分辨率MS/MS采集比当前最先进的技术快一个数量级。的MS 在本项目下开发的阵列技术将涉及离子回旋共振质谱法 这将产生更高的质量分辨能力、更高的质量测量精度以及更高的吞吐量 采集因此，在给定的实验期间可以鉴定一个数量级或更多的肽 这将极大地增加每个蛋白质组学分析的信息内容， 一系列可以研究的蛋白质。

PROJECT SUMMARY/ABSTRACT The study of human diseases at the molecular level has benefitted greatly by rapid advances in technology for genome sequencing. However, impediments for effective utilization of current and future genomic information relates to challenges associated with functional assignment of genes. The field of proteomics has arisen to help address this need, since proteins are the predominant functional molecules in cells. 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 wide 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 genes 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 high resolution 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.