A Research Resource for Ultra-sensitive High Throughput Proteomics

项目摘要--总体 高通量表型鉴定和分子表征技术的进展具有深远的意义 对生物研究的影响。然而，通常很难，例如，研究泛函 遗传变化的后果，因为缺乏适当的分析技术来定义功能 细胞状态。质谱学(MS)是目前最有效的分子定量方法 由基因表达改变引起的变化，但限制使其不能成为例行公事 广泛可用的工具。具体地说，蛋白质组学测量的灵敏度、吞吐量和精密度是 大大低于推动现代生物学重大进步的其他高通量技术 (例如测序和高含量成像)。在这次更新中，蛋白质组学资源的目标是广泛地 通过提供以下能力影响生物医学研究：获得至少10到100个- 折叠更小的样本，产生更全面的定量测量，提高低覆盖率 丰富的成分，如有问题的多肽异构体和翻译后修饰，并使 通过提供较大的测量增加来分析比目前实际大得多的样本集 吞吐量。我们将使用无损离子的新结构来解决蛋白质组学的主要缺陷 操作(超薄)离子迁移率(IM)-MS技术将极大地增加蛋白质组样本 并提供更好的重复性、灵敏度、量化的准确性和覆盖率 蛋白质组学测量。这些进展将得到快速纳升的创新方法的补充 规模化的机器人处理比目前可行的小得多的样本，并包括重要的分析 功能‘亚蛋白质组’(例如，磷酸蛋白质组学和基于活性的蛋白质组学样本)。机器人 样品处理将提供与SLIM IM-MS平台的速度匹配的吞吐量。这些进展 将与处理超薄IM-MS所需的算法和软件开发一起进行 平台数据流和生物信息学，用于从这些数据中获得生物洞察。总而言之，我们的 这项工作将通过将新功能应用于一组 具有挑战性的生物医学项目。事态发展将由两个机构向更广泛的社区传播。 常规方法(如讲习班和培训活动)和直接方法，包括直接致力于 将新技术“播种”给一些具有研发和开发专业知识的外部研究实验室 实施生物医学技术，并帮助促进从 跨多种MS平台类型的供应商数量。我们的项目将导致广泛和有影响力的初步 应用先进的蛋白质组学能力，并在年底前有效传播这些技术 最后一次资源的更新。

Project Summary – Overall Advances in high-throughput phenotyping and molecular characterization technologies have had profound impacts on biological research. However, it is frequently difficult, for example, to study the functional consequences of genetic changes because of the lack of suitable analytical technologies to define the functional cellular state. Mass spectrometry (MS) is presently the most powerful approach for quantifying the molecular changes that arises from altered gene expression, but limitations have prevented it from being a routine and broadly available tool. Specifically, the sensitivity, throughput and precision of proteomics measurements are significantly less than other high-throughput technologies that are driving major advances in modern biology (e.g. sequencing and high-content imaging). In this renewal the Proteomics Resource has the goal of broadly impacting biomedical research by providing the abilities to: obtain high quality data from at least 10- to 100- fold smaller samples, produce much more comprehensive quantitative measurements, improve coverage of low abundance components such as problematic peptide isomers and post translational modifications, and enable the analysis of far larger sample sets than presently practical by providing large increases in measurement throughput. We will address the key deficiencies of proteomics using new Structures for Lossless Ion Manipulations (SLIM) ion mobility (IM)-MS based technologies that will vastly increase proteomic sample throughput as well as provide improved reproducibility, sensitivity, accuracy of quantification and coverage of proteomics measurements. These advances will be complemented by innovative approaches for rapid nanoliter scale robotic processing of much smaller samples than presently feasible, and include analyses of important functional `sub-proteomes' (e.g. phosphoproteomics and activity-based proteomics samples). The robotic sample processing will provide throughput matching the speed of the SLIM IM-MS platform. These advances will occur in conjunction with algorithmic and software developments needed to handle the SLIM IM-MS platform data flow and bioinformatics for obtaining biological insights from these data. In combination, our work will lead to the rapid implementation of the new capabilities through their application to a set of challenging biomedical projects. The developments will be disseminated to the broader community by both conventional (e.g. workshops and training activities) and direct approaches that include working directly to `seed' the new technology in a number of outside research laboratories with expertise in developing and implementing biomedical technologies, and also helping to facilitate commercial implementations from a number of vendors spanning multiple MS platform types. Our project will result in broad and impactful initial applications of advanced proteomics capabilities, and effective dissemination of these technologies by the end of this final renewal of the Resource.