Proteome Capture in Hydrogel Beads for High Resolution Single Cell Analysis

摘要 在单细胞水平上对表面和细胞内蛋白质的分析在 免疫学、肿瘤学和诊断学。尽管已经出现了几个用于单细胞分析的平台， 由于其快速、灵敏和定量的特点，流式细胞术一直是最强大的技术之一。 使用目前的仪器，每个细胞可以测量20-40个蛋白质，每个细胞的速率为数千个 第二。然而，该平台的一个主要限制是已经被 开发并验证了流式细胞术。目前，只有不到2%的细胞内蛋白质靶标可以 通过流式细胞仪检测，而其他生化技术，如Western blotting，能够 测量超过25%。流式细胞术的这种限制很大程度上是由于细胞需要保持 完好无损，可供分析。这排除了完全变性蛋白质和蛋白质的样品制备方法 揭示被更广泛的抗体检测所需的表位。相反，在西区 印迹，蛋白质被变性并铺开在膜表面，使它们能够被 针对线性化表位产生的抗体。在这里，我们提出了一个技术平台 这会将单个细胞的蛋白质组转移到水凝胶珠中。这使得蛋白质可以变性。 完全经过严苛的变性处理，同时也将它们分散在太空中，以便更容易接触到 通过抗体试剂，因为珠子比原始细胞大。该系统的设计是为了保存 单细胞流式细胞术分析的关键优势，如表面染色的能力 复杂免疫细胞群体中的标志物、获取速度和检测灵敏度。这些珠子是 尺寸均匀，在溶液中稳定，光学透明，是高灵敏度的理想衬底 侦测。通过模拟通常在西方印迹中遇到的环境，我们预计 单细胞微珠平台将使数百到数千种额外的抗体试剂得以使用。这 现有试剂的巨大扩展将使下一代单细胞分析成为可能，直接分析 蛋白质而不是下游或上游的替代标记，信号的数量增加 可以监测的途径，并最终更深入地了解疾病状态和药物活性。

Summary Analysis of surface and intracellular proteins at the single cell level has become widespread in immunology, oncology, and diagnostics. Although several platforms have emerged for single cell analysis, flow cytometry has remained one of the most powerful due to its speed, sensitivity, and quantitative nature. With current instrumentation, 20-40 proteins can be measured per cell, at rates of thousands of cells per second. However, a major limitation of the platform is the number of antibody reagents that have been developed and validated for flow cytometry. Currently, less than 2% of intracellular protein targets can be measured by flow cytometry, while other biochemical techniques such as Western blotting are able to measure over 25%. This limitation in flow cytometry is largely due to the fact that the cells need to remain intact for analysis. This precludes sample preparation methods that would fully denature the proteins and expose the epitopes necessary for detection by a wider range of antibodies. Conversely, in Western blotting, the proteins are denatured and spread out on a membrane surface, making them accessible to antibodies which have been generated against linearized epitopes. Here we propose a technology platform that transfers the proteome of a single cell into a hydrogel bead. This enables the proteins to be denatured fully with harsh denaturation treatments, while also spreading them out in space to allow for easier access by antibody reagents, as the beads are larger than the original cells. The system is designed to preserve the critical advantages of single cell analysis by flow cytometry, such as the ability to stain for surface markers in complex immune cell populations, speed of acquisition, and detection sensitivity. The beads are also uniform in size, stable in solution, and optically clear, creating an ideal substrate for highly sensitive detection. By emulating the environment typically encountered in Western blots, we anticipate that the single cell bead platform will enable the use of hundreds to thousands of additional antibody reagents. This vast expansion in available reagents will enable next generation single cell analysis, the direct analysis of proteins instead of downstream or upstream surrogate markers, an increase in the number of signaling pathways that can be monitored, and ultimately deeper insight into disease states and drug activity.