High-throughput and high-efficiency sample preparation for single-cell proteomics using a nested nanowell chip

Nat Commun. 2021 Oct 29;12(1):6246. doi: 10.1038/s41467-021-26514-2.


Global quantification of protein abundances in single cells could provide direct information on cellular phenotypes and complement transcriptomics measurements. However, single-cell proteomics is still immature and confronts many technical challenges. Herein we describe a nested nanoPOTS (N2) chip to improve protein recovery, operation robustness, and processing throughput for isobaric-labeling-based scProteomics workflow. The N2 chip reduces reaction volume to <30 nL and increases capacity to >240 single cells on a single microchip. The tandem mass tag (TMT) pooling step is simplified by adding a microliter droplet on the nested nanowells to combine labeled single-cell samples. In the analysis of ~100 individual cells from three different cell lines, we demonstrate that the N2 chip-based scProteomics platform can robustly quantify ~1500 proteins and reveal membrane protein markers. Our analyses also reveal low protein abundance variations, suggesting the single-cell proteome profiles are highly stable for the cells cultured under identical conditions.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Animals
  • Biomarkers / analysis
  • Cell Line
  • Equipment Design
  • Lab-On-A-Chip Devices
  • Mice
  • Nanostructures / chemistry
  • Proteins / analysis
  • Proteomics / instrumentation*
  • Proteomics / methods*
  • RAW 264.7 Cells
  • Reproducibility of Results
  • Sequence Analysis, RNA
  • Single-Cell Analysis / instrumentation*
  • Single-Cell Analysis / methods*
  • Specimen Handling / instrumentation
  • Specimen Handling / methods
  • Tandem Mass Spectrometry / methods
  • Workflow


  • Biomarkers
  • Proteins