Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory, Richland, WA, 99354, USA.
Biological Sciences Division, Pacific Northwest National Laboratory, Richland, WA, 99354, USA.
Nat Commun. 2021 Oct 29;12(1):6246. doi: 10.1038/s41467-021-26514-2.
Global quantification of protein abundances in single cells could provide direct information on cellular phenotypes and complement transcriptomics measurements. However, single-cell proteomics is still immature and confronts many technical challenges. Herein we describe a nested nanoPOTS (N2) chip to improve protein recovery, operation robustness, and processing throughput for isobaric-labeling-based scProteomics workflow. The N2 chip reduces reaction volume to <30 nL and increases capacity to >240 single cells on a single microchip. The tandem mass tag (TMT) pooling step is simplified by adding a microliter droplet on the nested nanowells to combine labeled single-cell samples. In the analysis of ~100 individual cells from three different cell lines, we demonstrate that the N2 chip-based scProteomics platform can robustly quantify ~1500 proteins and reveal membrane protein markers. Our analyses also reveal low protein abundance variations, suggesting the single-cell proteome profiles are highly stable for the cells cultured under identical conditions.
单细胞内蛋白质丰度的全局定量分析可直接提供细胞表型信息，并与转录组学测量结果互补。然而，单细胞蛋白质组学仍处于起步阶段，面临许多技术挑战。本文中，我们描述了一种嵌套纳流控（N2）芯片，以提高基于等压标签的 scProteomics 工作流程中的蛋白质回收率、操作稳定性和处理通量。N2 芯片将反应体积减小到<30 nL，并将单个微芯片上的单细胞容量增加到>240 个。通过在嵌套纳米孔中添加微升液滴来简化串联质量标签（TMT）的池化步骤，从而将标记的单细胞样品进行合并。在对来自三种不同细胞系的约 100 个单个细胞的分析中，我们证明了基于 N2 芯片的 scProteomics 平台可以稳健地定量~1500 种蛋白质，并揭示膜蛋白标志物。我们的分析还表明蛋白质丰度变化较低，这表明在相同条件下培养的细胞中，单细胞蛋白质组谱高度稳定。
