Label-Free Profiling of up to 200 Single-Cell Proteomes per DayUsing a Dual-Column Nanoflow Liquid Chromatography Platform

Single-cell proteomics (SCP) has great potential toadvance biomedical research and personalized medicine. Thesensitivity of such measurements increases with low-flowseparations (<100 nL/min) due to improved ionization efficiency,but the time required for sample loading, column washing, andregeneration in these systems can lead to low measurementthroughput and inefficient utilization of the mass spectrometer.Herein, we developed a two-column liquid chromatography (LC)system that dramatically increases the throughput of label-free SCPusing two parallel subsystems to multiplex sample loading, onlinedesalting, analysis, and column regeneration. The integration ofMS1-based feature matching increased proteome coverage whenshort LC gradients were used. The high-throughput LC system was reproducible between the columns, with a 4% difference inmedian peptide abundance and a median CV of 18% across 100 replicate analyses of a single-cell-sized peptide standard. An averageof 621, 774, 952, and 1622 protein groups were identified with total analysis times of 7, 10, 15, and 30 min, corresponding to ameasurement throughput of 206, 144, 96, and 48 samples per day, respectively. When applied to single HeLa cells, we identifiednearly 1000 protein groups per cell using 30 min cycles and 660 protein groups per cell for 15 min cycles. We explored the possibilityof measuring cancer therapeutic targets with a pilot study comparing the K562 and Jurkat leukemia cell lines. This workdemonstrates the feasibility of high-throughput label-free single-cell proteomics.