Department of Chemistry & Biochemistry, University of Maryland, College Park, Maryland 20742, United States.
Anal Chem. 2024 Oct 22;96(42):16985-16993. doi: 10.1021/acs.analchem.4c04353. Epub 2024 Oct 9.
Current developments in single-cell mass spectrometry (MS) aim to deepen proteome coverage while enhancing analytical speed to study entire cell populations, one cell at a time. Custom-built microanalytical capillary electrophoresis (μCE) played a critical role in the foundation of discovery single-cell MS proteomics. However, requirements for manual operation, substantial expertise, and low measurement throughput have so far hindered μCE-based single-cell studies on large numbers of cells. Here, we design and construct a robotic capillary (RoboCap) platform that grants single-cell CE-MS with automation for proteomes limited to less than ∼100 nL. RoboCap remotely controls precision actuators to translate the sample to the fused silica separation capillary, using vials in this work. The platform is hermetically enclosed and actively pressurized to inject ∼1-250 nL of the sample into a CE separation capillary, with errors below ∼5% relative standard deviation (RSD). The platform and supporting equipment were operated and monitored remotely on a custom-written Virtual Instrument (LabView). Detection performance was validated empirically on ∼5-250 nL portions of the HeLa proteome digest using a trapped ion mobility mass spectrometer (timsTOF PRO). RoboCap improved CE-ESI sample utilization to ∼20% from ∼3% on the manual μCE, the closest reference technology. Proof-of-principle experiments found proteome identification and quantification to robustly return ∼1,800 proteins (∼13% RSD) from ∼20 ng of the HeLa proteome digest on this earlier-generation detector. RoboCap automates CE-MS for limited sample amounts, paving the way to electrophoresis-based high-throughput single-cell proteomics.
目前单细胞质谱（MS）的发展旨在提高分析速度，同时加深蛋白质组覆盖范围，以逐个细胞的方式研究整个细胞群体。定制的微分析毛细管电泳（μCE）在单细胞 MS 蛋白质组学的发现中发挥了关键作用。然而，手动操作、大量专业知识和低测量通量的要求迄今为止一直阻碍了基于 μCE 的大量细胞单细胞研究。在这里，我们设计并构建了一个机器人毛细管（RoboCap）平台，该平台可实现自动化单细胞 CE-MS，用于分析少于约 100 nL 的蛋白质组。RoboCap 通过远程控制精密执行器，将样品传输到融合硅分离毛细管中，在这项工作中使用小瓶。该平台是密封的，并主动加压，以将约 1-250 nL 的样品注入 CE 分离毛细管中，误差低于约 5%相对标准偏差（RSD）。该平台和配套设备由自定义编写的虚拟仪器（LabView）远程操作和监控。使用 trapped ion mobility mass spectrometer (timsTOF PRO) 在约 5-250 nL 的 HeLa 蛋白质组消化物部分上对检测性能进行了实证验证。与最接近的参考技术手动 μCE 相比，RoboCap 将 CE-ESI 样品利用率提高到约 20%，从约 3%提高到约 20%。原理验证实验发现，从较早一代探测器的约 20 ng HeLa 蛋白质组消化物中，该平台能够稳定地鉴定和定量约 1800 种蛋白质（约 13%RSD）。RoboCap 为有限的样品量自动化了 CE-MS，为基于电泳的高通量单细胞蛋白质组学铺平了道路。
