Proteome Capture in Hydrogel Beads for High Resolution Single Cell Analysis

总结 T细胞是最多样化的免疫细胞类型之一，并且是免疫缺陷病理学的核心。 一系列人类疾病。这种多样性在很大程度上是由它们的差异性决定的。 细胞表面蛋白质的表达，以及在较小程度上，细胞内蛋白质标记物的表达， 细胞仪虽然已经取得了重大进展，但由于缺乏 可用的抗体试剂。在已知的2万种人类蛋白质中，只有900种经过验证 细胞计数试剂，严重限制了可以测量的细胞表型和反应。 事实上，最近使用蛋白质组学（质谱法）和基因组学（scRNAseq）的研究 鉴定了数百种在T细胞亚群中差异表达的蛋白质， 这表明我们目前使用流式细胞术对T细胞的定义相当有限。但其他 基于抗体的方法，如Western印迹法，有超过一半的抗体可用。 蛋白质组在我们的第一阶段研究中，我们开发了允许流式细胞术的外切细胞方法 使用SDS和加热处理样品，类似于Western印迹样品。这种格式使得 流式细胞术样品适合用经验证可使用Western印迹法工作的抗体染色 污点。流式细胞术样品处理的这一根本性进展有望显着提高 增加可用于流式细胞术表征T细胞群的抗体数量 和细胞状态。在II期研究中，我们将进一步优化方法并进行含量测定 原代细胞样品的鉴定。使用优化的方法，我们将扩大现有的剧目 T细胞标志物减少了30%以上。最后，使用能够分析40个或更多个细胞的质谱细胞术， 每个细胞更多的参数，我们将创建一个包含25个已知表面标记的T细胞染色面板 和15种新发现的细胞内蛋白质。这个小组将是一个强大的工具， 新的T细胞亚群，用于在各种T细胞介导的疾病状态中发现生物标志物， 免疫肿瘤学治疗的分析。通过显著增加流式细胞仪的数量， 能够差异染色T细胞的试剂，我们寻求推动下一代的发现 以及它们在人类疾病进展和治疗功效中的作用。

Summary T cells are among the most diverse immunological cell types and are central to the pathology of a broad range of human diseases. This diversity has largely been defined by their differential expression of cell surface proteins and to a lesser extent, intracellular protein markers, using flow cytometry. Although significant progress has been made, the field is unnecessarily limited by the lack of available antibody reagents. Only 900 of the 20,000 known human proteins have validated flow cytometry reagents, severely limiting the cellular phenotypes and responses that can be measured. Indeed, recent studies using proteomics (mass spectrometry) and genomics (scRNAseq) have identified hundreds of proteins that appear to be differentially expressed within T cell subsets, suggesting that our current definition of T cells using flow cytometry is quite limited. However, other antibody-based methods such as Western blot have antibodies available for more than half the proteome. In our Phase I studies we developed the Exocells method that allows flow cytometry samples to be processed using SDS and heat, similar to Western blot samples. This format makes the flow cytometry samples amenable to staining with antibodies that are validated to work using Western blots. This fundamental advance in flow cytometry sample processing promises to dramatically increase the number of antibodies that can be used in flow cytometry to characterize T cell populations and cell states. In the Phase II studies, we will further optimize the method and perform assay qualification in primary cell samples. Using the optimized method, we will expand the existing repertoire of T cell markers by more than 30%. Finally, using mass cytometry, which is capable of analyzing 40 or more parameters per cell, we will create a T cell staining panel containing 25 known surface markers and 15 of the newly discovered intracellular proteins. This panel will be a powerful tool for identification of novel T cell subsets, for biomarker discovery in various T cell-mediated disease states, and for analysis of immuno-oncology therapeutics. By significantly increasing the number of flow cytometry reagents capable of differentially staining T cells, we seek to drive the discovery of the next generation of T cell subsets and their role in human disease progression and therapeutic efficacy.