Research Institute of Molecular Pathology (IMP), Vienna BioCenter (VBC), Vienna, Austria; Broad Institute of MIT and Harvard, Cambridge, Massachusetts, USA.
Research Institute of Molecular Pathology (IMP), Vienna BioCenter (VBC), Vienna, Austria; Cellenion SASU, Lyon, France.
Mol Cell Proteomics. 2023 Dec;22(12):100665. doi: 10.1016/j.mcpro.2023.100665. Epub 2023 Oct 14.
Multiplexed and label-free mass spectrometry-based approaches with single-cell resolution have attributed surprising heterogeneity to presumed homogenous cell populations. Even though specialized experimental designs and instrumentation have demonstrated remarkable advances, the efficient sample preparation of single cells still lags. Here, we introduce the proteoCHIP, a universal option for single-cell proteomics sample preparation including multiplexed labeling up to 16-plex with high sensitivity and throughput. The automated processing using a commercial system combining single-cell isolation and picoliter dispensing, the cellenONE, reduces final sample volumes to low nanoliters submerged in a hexadecane layer simultaneously eliminating error-prone manual sample handling and overcoming evaporation. The specialized proteoCHIP design allows direct injection of single cells via a standard autosampler resulting in around 1500 protein groups per TMT10-plex with reduced or eliminated need for a carrier proteome. We evaluated the effect of wider precursor isolation windows at single-cell input levels and found that using 2 Da isolation windows increased overall sensitivity without significantly impacting interference. Using the dedicated mass spectrometry acquisition strategies detailed here, we identified on average close to 2000 proteins per TMT10-plex across 170 multiplexed single cells that readily distinguished human cell types. Overall, our workflow combines highly efficient sample preparation, chromatographic and ion mobility-based filtering, rapid wide-window data-dependent acquisition analysis, and intelligent data analysis for optimal multiplexed single-cell proteomics. This versatile and automated proteoCHIP-based sample preparation approach is sufficiently sensitive to drive biological applications of single-cell proteomics and can be readily adopted by proteomics laboratories.
基于多重无标记质谱的单细胞分辨率方法使原本均匀的细胞群体表现出惊人的异质性。尽管专门的实验设计和仪器已经取得了显著的进展，但单细胞的有效样品制备仍然滞后。在这里，我们介绍了 proteoCHIP，这是一种通用的单细胞蛋白质组学样品制备选项，包括多达 16 重的多重标记，具有高灵敏度和高通量。使用商业系统（结合单细胞分离和皮升级分配的 cellenONE）进行自动化处理，将最终样品体积减少到低纳升级，同时浸没在十六烷层中，从而消除了易错的手动样品处理并克服了蒸发。专门的 proteoCHIP 设计允许通过标准自动进样器直接注射单细胞，从而在每个 TMT10 多重实验中获得约 1500 个蛋白质组，减少或消除了对载体蛋白质组的需求。我们评估了在单细胞输入水平下更宽的前体分离窗口的效果，发现使用 2 Da 分离窗口可以在不显著影响干扰的情况下提高整体灵敏度。使用这里详细介绍的专用质谱采集策略，我们在 170 个多重化的单细胞中平均鉴定出每个 TMT10 多重实验近 2000 个蛋白质，这些蛋白质可以轻松区分人类细胞类型。总体而言，我们的工作流程结合了高效的样品制备、基于色谱和离子淌度的过滤、快速宽窗口数据依赖采集分析以及智能数据分析，以实现最佳的多重化单细胞蛋白质组学。这种基于 proteoCHIP 的多功能和自动化样品制备方法非常灵敏，可以推动单细胞蛋白质组学的生物学应用，并且可以轻松被蛋白质组学实验室采用。
