A Research Resource for Ultra-sensitive High Throughput Proteomics

项目概要-总体 高通量表型分析和分子表征技术的进步对人类的免疫功能产生了深远的影响。 对生物学研究的影响。然而，它往往是困难的，例如，研究的功能， 由于缺乏合适的分析技术来确定功能性基因， 细胞状态质谱法（MS）是目前用于定量分子生物学的最有力的方法。 基因表达改变引起的变化，但限制使其无法成为常规， 广泛使用的工具。具体而言，蛋白质组学测量的灵敏度、通量和精度是 远低于其他推动现代生物学重大进步的高通量技术 (e.g.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 通过提供以下能力来影响生物医学研究：从至少10到100个国家获得高质量的数据， 倍更小的样本，产生更全面的定量测量，提高低覆盖率 丰度组分，如有问题的肽异构体和翻译后修饰，并使 通过提供测量的大幅增加，分析比目前实际情况大得多的样本集 吞吐量我们将使用新的无损离子结构来解决蛋白质组学的关键缺陷。 操作（SLIM）离子迁移率（IM）-基于MS的技术，将大大增加蛋白质组学样本 通量以及提供改进的再现性、灵敏度、定量的准确性和覆盖率。 蛋白质组学测量。这些进展将得到创新方法的补充， 规模比目前可行的小得多的样品的机器人处理，并包括重要的分析， 功能性“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品）。机器人 样品处理将提供与SLIM IM-MS平台的速度相匹配的吞吐量。这些进步 将与处理SLIM 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学见解。结合起来，我们的 这项工作将通过将新能力应用于一系列 具有挑战性的生物医学项目。这些发展将由双方向更广泛的社区传播， 传统的（例如讲习班和培训活动）和直接的办法，包括直接努力， 将新技术“播种”到一些具有开发和 实施生物医学技术，并帮助促进商业实施， 跨多种MS平台类型的供应商数量。我们的项目将产生广泛和有影响力的初步成果， 先进的蛋白质组学能力的应用，以及这些技术的有效传播， 资源的最终更新

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.