An automated nanowell-array workflow for quantitative multiplexed single-cell proteomics sample preparation at high sensitivity

Mol Cell Proteomics. 2023 Oct 13:100665. doi: 10.1016/j.mcpro.2023.100665. Online ahead of print.ABSTRACTMultiplexed and label-free mass spectrometry-based approaches with single-cell resolution have attributed surprising heterogeneity to presumed homogenous cell populations. Even though specialized experimental designs and instrumentation have demonstrated remarkable advances, the efficient sample preparation of single cells still lags. Here, we introduce the proteoCHIP, a universal option for single-cell proteomics sample preparation including multiplexed labeling up to 16-plex with high sensitivity and throughput. The automated processing using a commercial system combining single-cell isolation and picoliter dispensing, the cellenONE®, reduces final sample volumes to low nanoliters submerged in a hexadecane layer simultaneously eliminating error-prone manual sample handling and overcoming evaporation. The specialized proteoCHIP design allows direct injection of single cells via a standard autosampler resulting in around 1,500 protein groups per TMT10-plex with reduced or eliminated need for a carrier proteome. We evaluated the effect of wider precursor isolation windows at single-cell input levels and found that using 2 Da isolation windows increased overall sensitivity without significantly impacting interference. Using the dedicated MS acquisition strategies detailed here, we identified on average close to 2,000 proteins per TMT10-plex across 170 multiplexed single cel...
Source: Molecular and Cellular Proteomics : MCP - Category: Molecular Biology Authors: Source Type: research