Institute of Chemistry, Academia Sinica, Taipei, 11529, Taiwan.
Nano Science and Technology Program, Taiwan International Graduate Program, Academia Sinica, Taipei, 11529, Taiwan.
Nat Commun. 2022 Jan 10;13(1):37. doi: 10.1038/s41467-021-27778-4.
Single-cell proteomics can reveal cellular phenotypic heterogeneity and cell-specific functional networks underlying biological processes. Here, we present a streamlined workflow combining microfluidic chips for all-in-one proteomic sample preparation and data-independent acquisition (DIA) mass spectrometry (MS) for proteomic analysis down to the single-cell level. The proteomics chips enable multiplexed and automated cell isolation/counting/imaging and sample processing in a single device. Combining chip-based sample handling with DIA-MS using project-specific mass spectral libraries, we profile on average ~1,500 protein groups across 20 single mammalian cells. Applying the chip-DIA workflow to profile the proteomes of adherent and non-adherent malignant cells, we cover a dynamic range of 5 orders of magnitude with good reproducibility and <16% missing values between runs. Taken together, the chip-DIA workflow offers all-in-one cell characterization, analytical sensitivity and robustness, and the option to add additional functionalities in the future, thus providing a basis for advanced single-cell proteomics applications.
单细胞蛋白质组学可以揭示细胞表型异质性和生物学过程中特定于细胞的功能网络。在这里，我们提出了一种简化的工作流程，将用于一体式蛋白质组样品制备的微流控芯片与用于蛋白质组分析的数据非依赖性采集 (DIA) 质谱 (MS) 相结合，可将蛋白质组分析深入到单细胞水平。该蛋白质组芯片可在单个设备中实现复份和自动化的细胞分离/计数/成像和样品处理。通过使用特定于项目的质谱文库，将基于芯片的样品处理与 DIA-MS 相结合，我们平均可以对 20 个单个哺乳动物细胞中的约 1500 个蛋白质组进行分析。将芯片-DIA 工作流程应用于分析贴壁和非贴壁恶性细胞的蛋白质组，我们可以在 5 个数量级的动态范围内进行覆盖，具有良好的重现性和<16%的运行间缺失值。总之，芯片-DIA 工作流程提供了一体式细胞特征分析、分析灵敏度和稳健性，并提供了未来添加其他功能的选项，从而为高级单细胞蛋白质组学应用提供了基础。
