Development\nand Validation of RoboCap, a Robotic Capillary\nPlatform to Automate Capillary Electrophoresis Mass Spectrometry En\nRoute to High-Throughput Single-Cell Proteomics

Current developments in single-cell\nmass spectrometry\n(MS) aim\nto deepen proteome coverage while enhancing analytical speed to study\nentire cell populations, one cell at a time. Custom-built microanalytical\ncapillary electrophoresis (μCE) played a critical role in the\nfoundation of discovery single-cell MS proteomics. However, requirements\nfor manual operation, substantial expertise, and low measurement throughput\nhave so far hindered μCE-based single-cell studies on large\nnumbers of cells. Here, we design and construct a robotic capillary\n(RoboCap) platform that grants single-cell CE-MS with automation for\nproteomes limited to less than ∼100 nL. RoboCap remotely controls\nprecision actuators to translate the sample to the fused silica separation\ncapillary, using vials in this work. The platform is hermetically\nenclosed and actively pressurized to inject ∼1–250 nL\nof the sample into a CE separation capillary, with errors below ∼5%\nrelative standard deviation (RSD). The platform and supporting equipment\nwere operated and monitored remotely on a custom-written Virtual Instrument\n(LabView). Detection performance was validated empirically on ∼5–250\nnL portions of the HeLa proteome digest using a trapped ion mobility\nmass spectrometer (timsTOF PRO). RoboCap improved CE-ESI sample utilization\nto ∼20% from ∼3% on the manual μCE, the closest\nreference technology. Proof-of-principle experiments found proteome\nidentification and quantification to robustly return ∼1,800\nproteins (∼13% RSD) from ∼20 ng of the HeLa proteome\ndigest on this earlier-generation detector. RoboCap automates CE-MS\nfor limited sample amounts, paving the way to electrophoresis-based\nhigh-throughput single-cell proteomics.