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

Single-cell
proteomics (SCP) has great potential to advance biomedical
research and personalized medicine. The sensitivity of such measurements
increases with low-flow separations (<100 nL/min) due to improved
ionization efficiency, but the time required for sample loading, column
washing, and regeneration in these systems can lead to low measurement
throughput 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 SCP using two parallel subsystems
to multiplex sample loading, online desalting, analysis, and column
regeneration. The integration of MS1-based feature matching increased
proteome coverage when short LC gradients were used. The high-throughput
LC system was reproducible between the columns, with a 4% difference
in median peptide abundance and a median CV of 18% across 100 replicate
analyses of a single-cell-sized peptide standard. An average of 621,
774, 952, and 1622 protein groups were identified with total analysis
times of 7, 10, 15, and 30 min, corresponding to a measurement throughput
of 206, 144, 96, and 48 samples per day, respectively. When applied
to single HeLa cells, we identified nearly 1000 protein groups per
cell using 30 min cycles and 660 protein groups per cell for 15 min
cycles. We explored the possibility of measuring cancer therapeutic
targets with a pilot study comparing the K562 and Jurkat leukemia
cell lines. This work demonstrates the feasibility of high-throughput
label-free single-cell proteomics.