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

Abstract

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.

Parallelization with Dual-Trap Single-Column Configuration Maximizes Throughput of Proteomic Analysis

“…, Ultimate 3000 nano-RSLC and Waters Acuity) are widely implemented making DTSC accessible to many researchers. The dual analytical column, also referred to as the double barrel strategy, may use a valve to select which column is connected to the electrospray source during analyte elution at high flowrates, but this post-column volume reduces performance at the nano- and low microliter/min flowrates, thus a custom electrospray source with two separate emitters is required . Furthermore, a second analytical column is an additional source of variability.…”

In-Depth Mass Spectrometry-Based Proteomics of Formalin-Fixed, Paraffin-Embedded Tissues with a Spatial Resolution of 50–200 μm

“…Using the single-pot, solid-phase-enhanced (SP3) sample processing method, approximately 1500 proteins were identified from 10 μm thick brain tissue sections with a 0.06 mm 2 area . Our group has also focused on improving the sensitivity of MS-based proteomics for low-input samples including single cells − and trace tissue samples − through improvements in sample preparation, separations, ionization, and mass spectrometry. To minimize losses during sample preparation, we developed nanodroplet processing in one pot for trace samples (nanoPOTS), which miniaturizes processing volumes to the nanoliter scale to minimize surface exposure and transfer losses while enhancing reaction kinetics.…”

Parallelization with Dual-Trap Single-Column Configuration Maximizes Throughput of Proteomic Analysis

“…Ultimate 3000 nano-RSLC and Waters Acuity) are widely implemented making this strategy accessible to many researchers. The dual column strategy can use a valve to select the column that is connected to the electrospray source during analyte elution[43], however this post-column volume reduces performance at the nano- and low micro-liter/min flowrates and a custom electrospray source that can accommodate two separate emitters is required [44]. Furthermore, a second analytical column is an additional source of variability.…”