Area A: In-Depth Proteome Mapping of the Tumor Microenvironment with Single- Cell Resolution

A 区：单细胞分辨率的肿瘤微环境深度蛋白质组图谱

• 批准号: 9752092
• 负责人: Ryan T Kelly
• 金额: $ 106.96万
• 依托单位: BRIGHAM YOUNG UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-09-30 至 2021-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9752092
• 关键词: Area; Depth; Proteome; Mapping; Tumor

PROJECT SUMMARY/ABSTRACT The development of effective therapies for cancer requires a deep molecular understanding of tumor heterogeneity by advanced omics technologies with spatially resolved measurements. Unfortunately, there are substantial gaps in existing capabilities in terms of sensitivity. For example, a minimum of many thousands of cells is required for in-depth profiling of proteins in a biological sample. We have recently developed a breakthrough technology, termed nanoPOTS (Nanodroplet Preparation in One pot for Trace Samples) which, when coupled to ultrasensitive liquid chromatography-mass spectrometry (LC-MS), enables effective analysis of as few as 10 mammalian cells with a coverage of >3000 identified proteins. We hypothesize that the nanoPOTS platform will be an enabling technology to characterize the molecular underpinnings of tumor heterogeneity by creating 3D proteome maps of human tumors. The overall objective of our study is to extend and validate this analysis platform to enable single-cell resolution measurements at high throughput (>100 samples per day) and apply the platform to create 3D proteome maps of human tumors. Studies in Aim 1 will focus on optimizing and validating the ultrasensitive nanoPOTS proteomic workflow to enable robust proteome profiling of ≥3,000 protein groups from single human cells obtained by both flow cytometry and laser capture microdissection (LCM). Aim 2 will evaluate and compare two complementary technologies for increasing measurement throughput to ~100 cells/day with minimal impact on proteome coverage. We will determine whether multiplexing through application of isobaric labels for pooled analysis, or rapid LC coupled with ultrahigh resolution ion mobility spectrometry-MS provides greatest coverage, quantitation and reproducibility for single cell proteomics at the desired throughput. In Aim 3, we will apply the optimized platform to create in-depth proteome maps for human pancreatic ductal adenocarcinomas (PDAs) at single cell resolution. Following initial targeted studies of specific cells of interest within sectioned tissues, we will use a combination of cryosectioning, LCM and the optimized nanoPOTS platform to analyze single cells within the tumor microenvironment. 3D reconstruction of these in-depth, spatially resolved proteomic analyses will provide the first global proteomic tumor maps at single-cell resolution. This project will not only establish an innovative measurement capability that will broadly advance cancer research, but will also provide unique molecular insights into cellular heterogeneity relevant to PDA pathology. The resulting platform will be disseminated through a combination of publication and commercialization.

项目概要/摘要 开发有效的癌症疗法需要对肿瘤有深入的分子了解 不幸的是，先进的组学技术和空间分辨测量存在异质性。 现有能力在灵敏度方面存在巨大差距，例如，至少有数千个。 深入分析生物样品中的蛋白质需要细胞。我们最近开发了一种方法。 突破性技术，称为 nanoPOTS（微量样品一锅纳米液滴制备）， 当与超灵敏液相色谱-质谱 (LC-MS) 结合使用时，可以有效分析 少至 10 个哺乳动物细胞，覆盖超过 3000 个已识别蛋白质。 该平台将成为一种通过以下方式表征肿瘤异质性的分子基础的使能技术： 创建人类肿瘤的 3D 蛋白质组图谱 我们研究的总体目标是扩展和验证这一点。 分析平台，可实现高通量（每天 > 100 个样本）的单细胞分辨率测量 应用该平台创建人类肿瘤的 3D 蛋白质组图谱 目标 1 的研究将侧重于优化和分析。 验证超灵敏的 nanoPOTS 蛋白质组工作流程，以实现 ≥3,000 种蛋白质的稳健蛋白质组分析 通过流式细胞术和激光捕获显微切割（LCM）获得的单个人类细胞组。 2 将评估和比较两种互补技术，以将测量吞吐量提高到约 100 细胞/天，对蛋白质组覆盖率的影响最小。我们将确定是否通过应用进行复用。 用于混合分析的同量异位标记，或快速 LC 与超高分辨率离子迁移谱-MS 联用 以所需的通量为单细胞蛋白质组学提供最大的覆盖范围、定量和重现性。 在目标3中，我们将应用优化的平台为人类胰腺导管创建深入的蛋白质组图谱 在对感兴趣的特定细胞进行初步靶向研究后，进行单细胞分辨率的腺癌（PDA）。 在切片组织中，我们将结合使用冷冻切片、LCM 和优化的 nanoPOTS 分析肿瘤微环境中的单细胞的平台，对这些进行深入的空间重建。 解析的蛋白质组分析将提供第一个单细胞分辨率的全球蛋白质组肿瘤图谱。 该项目不仅将建立一种创新的测量能力，广泛推进癌症研究， 但也将为与 PDA 病理学相关的细胞异质性提供独特的分子见解。 由此产生的平台将通过出版和商业化相结合的方式进行传播。

项目成果

Ultrasensitive single-cell proteomics workflow identifies >1000 protein groups per mammalian cell.

• DOI: 10.1039/d0sc03636f
• 发表时间: 2020-11-17
• 期刊: Chemical science
• 影响因子: 8.4
• 作者: Cong Y;Motamedchaboki K;Misal SA;Liang Y;Guise AJ;Truong T;Huguet R;Plowey ED;Zhu Y;Lopez-Ferrer D;Kelly RT
• 通讯作者: Kelly RT

Fully Automated Sample Processing and Analysis Workflow for Low-Input Proteome Profiling.

• DOI: 10.1021/acs.analchem.0c04240
• 发表时间: 2021-01-26
• 期刊: Analytical chemistry
• 影响因子: 7.4
• 作者: Liang Y;Acor H;McCown MA;Nwosu AJ;Boekweg H;Axtell NB;Truong T;Cong Y;Payne SH;Kelly RT
• 通讯作者: Kelly RT

Improved Single-Cell Proteome Coverage Using Narrow-Bore Packed NanoLC Columns and Ultrasensitive Mass Spectrometry.

• DOI: 10.1021/acs.analchem.9b04631
• 发表时间: 2020-02-04
• 期刊: Analytical chemistry
• 影响因子: 7.4
• 作者: Cong Y;Liang Y;Motamedchaboki K;Huguet R;Truong T;Zhao R;Shen Y;Lopez-Ferrer D;Zhu Y;Kelly RT
• 通讯作者: Kelly RT

Adapting a Low-Cost and Open-Source Commercial Pipetting Robot for Nanoliter Liquid Handling.

• DOI: 10.1177/2472630320973591
• 发表时间: 2021-06
• 期刊: SLAS technology
• 影响因子: 2.7
• 作者: Councill EEAW;Axtell NB;Truong T;Liang Y;Aposhian AL;Webber KGI;Zhu Y;Cong Y;Carson RH;Kelly RT
• 通讯作者: Kelly RT

In-Depth Mass Spectrometry-Based Proteomics of Formalin-Fixed, Paraffin-Embedded Tissues with a Spatial Resolution of 50-200 μm.

• DOI: 10.1021/acs.jproteome.2c00409
• 发表时间: 2022-09-02
• 期刊: JOURNAL OF PROTEOME RESEARCH
• 影响因子: 4.4
• 作者: Nwosu, Andikan J.;Misal, Santosh A.;Thy Truong;Carson, Richard H.;Webber, Kei G., I;Axtell, Nathaniel B.;Liang, Yiran;Johnston, S. Madisyn;Virgin, Kenneth L.;Smith, Ethan G.;Thomas, George, V;Morgan, Terry;Price, John C.;Kelly, Ryan T.
• 通讯作者: Kelly, Ryan T.