Massive single cell proteomics for cancer biology

项目摘要/摘要 单细胞技术已经成为生物医学和细胞生物学研究的基石。下一步- 基于世代测序的技术使得转录表达的大规模表征成为可能 在临床标本的单个细胞中发现了与发病机制相关的意外的细胞异质性。 然而，许多综合研究表明，在丰富的空气中 RNA转录本及其相应的蛋白质，是细胞表型的主要决定因素。我们假设 基于质谱仪的单细胞蛋白质组学可以提供对细胞异质性的直接洞察和 告知与疾病进展和治疗抵抗有关的蛋白质标记物。这样做的总体目标是 该项目是开发一个高通量的单细胞蛋白质组学(ScProtetics)平台，以使常规 以经济高效的方式分析2000种蛋白质深度下的10,000个单细胞。已开发的技术 将通过与商业伙伴的密切合作向研究界传播。我们会 同时应用scProtetics研究恶性浆细胞和免疫细胞的异质性 多发性骨髓瘤患者的人群。我们将通过三个具体目标实现这些目标：1) 结合增强型多路复用法建立超高通量单细胞制备方法 拥有高密度嵌套纳米POTS芯片和多通道液滴分配系统；我们的目标是处理 单个微芯片中的&gt；2000个细胞，以及用于单个LC-MS分析的多个标签36个单个细胞；2) 提高LC-MS系统的吞吐量、灵敏度和定量准确度。一种双柱纳米LC 系统和基于FAIMS的MS采集方法将被开发，以实现对每个 3)应用单链蛋白质组学技术对10,000个血浆和免疫细胞进行分析 来自多发性骨髓瘤患者。我们将把sc蛋白质组学与现有的scRNA-seq数据相结合来探索肿瘤 异质性、嵌合抗原受体T细胞(CAR-T)标记和免疫微环境 多发性骨髓瘤。这项研究具有很高的创新性，因为提出的单细胞蛋白质组学平台将 成为同类产品中第一个常规且可靠地表征10,000个单细胞的产品，其吞吐量可与 单细胞转录组。这也是首次对从人脑组织分离的原代液体肿瘤细胞进行scProtetics研究。 病理环境，如多发性骨髓瘤患者的骨髓。影响声明：肿瘤异质性具有 在癌症进化、肿瘤空间组织和临床治疗中不可或缺的含义。单细胞 蛋白质组学可以为解开这些复杂的关系并阐明其机制提供基础。 癌症进展和对治疗方法的亚克隆抵抗。

PROJECT SUMMARY/ABSTRACT Single-cell technologies have become the cornerstone of biomedical and cell biology research. Next- generation sequencing-based technologies have enabled large-scale characterization of transcript expressions in single cells from clinical specimens and reveal unexpected cellular heterogeneity related to pathogenesis. However, many integrative studies have shown only low to moderate correlations between the abundance of RNA transcripts and their corresponding proteins, the main determinants of cell phenotype. We hypothesize mass spectrometry-based single-cell proteomics could provide direct insight on the cellular heterogeneity and inform protein markers related to disease progression and resistance to therapy. The overall objective of this project is to develop a high throughput single-cell proteomics (scProteomics) platform to enable the routine analysis of >10,000 single cells at a depth of 2000 proteins in a cost-efficient way. The developed technology will be disseminated to the research community through close collaboration with a commercial partner. We will also apply scProteomics to interrogate the heterogeneity of both malignant plasma cell and immune cell populations from multiple myeloma patients. We will pursue these goals through three specific aims: 1) To establish an ultra-high throughput single-cell preparation method by coupling an enhanced multiplexing method with high-density nested nanoPOTS chips and multi-channel droplet dispensing system; We aim to process >2000 cells in a single microchip, and multiplex-label 36 single cells for a single LC-MS analysis; 2) To advance the throughput, sensitivity, and quantitation accuracy of LC-MS system. A dual-column nanoLC system and a FAIMS-based MS acquisition method will be developed to enable the analysis of >860 cells per day with high quantitation precision; 3) To apply scProteomics to profile ~10,000 plasma and immune cells from MM patients. We will integrate scProteomics with existing scRNA-seq data to explore tumor heterogeneity, chimeric antigen receptor T-cells (CAR-T) markers, and the immune microenvironment in multiple myeloma. This research is highly innovative because the proposed single-cell proteomics platform will be the first of its kind to routinely and reliably characterize > 10,000 single cells at a throughput comparable to single-cell transcriptomics. It is also the first scProteomics study of primary liquid tumor cells isolated from the pathological environment, e.g. bone marrow of MM patients. Statement of Impact: Tumor heterogeneity has indispensable implications in cancer evolution, tumoral spatial organization, and clinical treatment. Single-cell proteomics could provide a basis to unravel these complicated relationships and to clarify the mechanisms of cancer progression and subclone resistance to therapeutic treatments.