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Abstract：
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.
摘要：
具有单细胞分辨率的基于多重和无标记质谱的方法将令人惊讶的异质性归因于假定的同源细胞群体。尽管专门的实验设计和仪器已经证明了显著的进步，单细胞的有效样品制备仍然滞后。这里,我们介绍了proteochip,单细胞蛋白质组学样品制备的通用选择，包括高灵敏度和高通量的高达16-plex的多重标记。使用结合单细胞分离和皮升分配的商业系统的自动化处理，celenONE®,将最终样品体积减少到浸没在十六烷层中的低纳升，同时消除了容易出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，导致每个TMT10-plex约1,500个蛋白质组，减少或消除了对载体蛋白质组的需要。我们评估了在单细胞输入水平下更宽的前体隔离窗口的效果，发现使用2Da隔离窗口增加了整体灵敏度，而不会显着影响干扰。使用此处详述的专用MS获取策略，我们在170个易于区分人类细胞类型的多重单细胞中,每个TMT10-plex平均鉴定出接近2,000种蛋白质.总的来说,我们的工作流程结合了高效的样品制备，色谱和基于离子迁移率的过滤，快速宽窗口DDA分析和智能数据分析，以实现最佳的多重单细胞蛋白质组学。这种多功能和自动化的基于proteochip的样品制备方法对于驱动单细胞蛋白质组学的生物学应用是足够敏感的,并且可以容易地被蛋白质组学实验室采用。
