Advanced Sample Preparation, Separation and Multiplexed Analysis for In-Depth Proteome Profiling of >1000 Single Cells Per Day

先进的样品制备、分离和多重分析，每天对超过 1000 个单细胞进行深入的蛋白质组分析

基本信息

批准号：
10642310
负责人：
Ryan T Kelly
金额：
$ 53.04万
依托单位：
BRIGHAM YOUNG UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-08-01 至 2026-07-31
项目状态：
未结题

项目摘要

PROJECT SUMMARY/ABSTRACT Cancer tissues exhibit a high degree of phenotypic heterogeneity and plasticity and contain numerous subpopulations of cells in various states. Quantifying this heterogeneity at the single-cell level and with molecular depth across large numbers of cells provides information that cannot be obtained at the bulk scale and will ultimately lead to improved diagnostics and more effective treatments. While single-cell nucleic acid sequencing approaches are having a significant impact on cancer research, proteins mediate the bulk of cellular function and are the targets of most therapeutics. There is thus an urgent need to develop new technologies for large- scale direct proteome profiling at the single-cell level. To fill this gap, mass spectrometry (MS)-based profiling of protein expression in single cells has recently been demonstrated through the implementation of more efficient sample processing workflows, novel experimental designs and improved instrument sensitivity. Label-free MS- based proteomics can now quantify >2,000 protein groups per cell across >4 orders of magnitude of dynamic range, but efforts to profile more than a few dozen cells per day have resulted in significantly reduced proteome coverage. This low throughput is insufficient for the large-scale statistically powered studies required to characterize heterogeneity in cancer cell populations. To increase measurement throughput, multiplexed workflows based on isobaric tandem mass tags (TMTs) enable up to 18 single cells to be measured in an LC- MS analysis, but these have still been limited to ~100 cells/day and, as generally implemented, suffer from a large proportion of missing values and other issues affecting quantitative performance. Our overall objective is to develop a platform that combines simplified pipette-free high-throughput sample preparation with rapid, multicolumn liquid chromatography separations and ‘greedy’ data-dependent acquisition to profile >2000 proteins per cell with a measurement throughput of >1000 single cells per day. We hypothesize that the advanced sample preparation and separation, combined with a far more efficient MS acquisition workflow, will achieve in-depth SCP with a 10× throughput gain, thus providing a capability for direct, in-depth and large-scale protein quantification that is analogous to single-cell RNA-seq. Studies in Aim 1 will focus on developing massively parallel centrifugal nanoliter dispensing to prepare >10,000 single-cells per day at a total reagent and consumables cost of <$0.40/cell. In Aim 2, we will develop rapid, robust and high-peak-capacity 20-min nanoLC separations with 100% duty cycle. In Aim 3, we will develop a novel ‘greedy’ data acquisition strategy in which only proteotypic peptides are selected for fragmentation, and with custom automatic gain control settings and fragmentation energy for each peptide, providing an unprecedented combination of sensitivity and throughput. With this next-generation platform, we will profile >10,000 cells to study acquired resistance to autophagy inhibitors in the context of autophagy-dependent triple negative breast cancer, thus establishing an innovative platform for advancing biomedical research and individualizing therapy.

项目概要/摘要癌组织表现出高度的表型异质性和可塑性，并含有大量的在单细胞水平和分子水平上量化这种异质性。大量细胞的深度提供了无法大规模获得的信息，并且将最终导致改进的诊断和更有效的治疗，而单细胞核酸测序。方法对癌症研究产生重大影响，蛋白质介导大部分细胞功能因此，迫切需要开发用于大规模治疗的新技术。在单细胞水平上进行直接蛋白质组分析为了填补这一空白，基于质谱 (MS) 的分析得到了应用。最近通过实施更有效的方法证明了单细胞中的蛋白质表达样品处理工作流程、新颖的实验设计和改进的无标记 MS-仪器灵敏度。基于蛋白质组学现在可以量化每个细胞 > 2,000 个蛋白质组，动态范围 > 4 个数量级范围，但每天分析数十个以上细胞的努力导致蛋白质组显着减少这种低通量不足以满足大规模研究人员进行研究所需的需求。为了提高测量通量，进行多重分析。基于同量异位串联质量标签 (TMT) 的工作流程可在 LC 中测量多达 18 个单细胞 MS 分析，但这些分析仍限于约 100 个细胞/天，并且按照普遍实施的方式，会受到大比例的缺失值和其他影响定量性能的问题是我们的总体目标。开发一个平台，将简化的无移液器高通量样品制备与快速、多柱液相色谱分离和“贪婪”数据依赖性采集以分析 >2000 每个细胞的蛋白质，每天的测量吞吐量 > 1000 个单细胞。先进的样品制备和分离，结合更高效的 MS 采集工作流程，将实现深度SCP，吞吐量提升10倍，提供直接、深入、大规模的能力目标 1 中的研究将侧重于开发类似于单细胞 RNA 测序的蛋白质定量。大规模并行离心纳升分配，每天可在总试剂中制备 >10,000 个单细胞，耗材成本<0.40 美元/单元在目标 2 中，我们将开发快速、稳定且高峰值容量的 20 分钟 nanoLC。在目标 3 中，我们将开发一种新颖的“贪婪”数据采集策略，其中仅选择蛋白型肽进行片段化，并具有自定义自动增益控制设置和每个肽的裂解能量，提供前所未有的灵敏度和通量组合。借助这个下一代平台，我们将分析超过 10,000 个细胞，以研究获得性自噬抵抗抑制剂在自噬依赖性三阴性乳腺癌的背景下，从而建立了一个创新的推进生物医学研究和个体化治疗的平台。