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

Jongmin Woo1, Sarah M Williams1, Lye Meng Markillie1

  • 1Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory, Richland, WA, 99354, USA.

Nature Communications
|October 30, 2021
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Summary
This summary is machine-generated.

A new nested nanoPOTS (N2) chip enhances single-cell proteomics by improving protein recovery and throughput. This technology robustly quantifies thousands of proteins, offering insights into cellular phenotypes.

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Area of Science:

  • Biochemistry
  • Cell Biology
  • Proteomics

Background:

  • Single-cell proteomics offers valuable insights into cellular phenotypes and complements transcriptomics.
  • Current single-cell proteomics methods face technical challenges in protein recovery, robustness, and throughput.

Purpose of the Study:

  • To develop an improved nested nanoPOTS (N2) chip for enhanced single-cell proteomics.
  • To increase protein recovery, operational robustness, and processing throughput in scProteomics workflows.

Main Methods:

  • Utilized a nested nanoPOTS (N2) chip with reduced reaction volumes (<30 nL) and increased capacity (>240 cells/chip).
  • Simplified tandem mass tag (TMT) pooling by adding microliter droplets on nested nanowells.
  • Analyzed approximately 100 individual cells from three distinct cell lines.

Main Results:

  • The N2 chip-based platform robustly quantified approximately 1500 proteins per cell.
  • Successfully identified membrane protein markers.
  • Observed low variation in protein abundance, indicating stable single-cell proteome profiles under identical conditions.

Conclusions:

  • The N2 chip significantly advances single-cell proteomics capabilities.
  • This technology enables robust quantification of a substantial number of proteins at the single-cell level.
  • Demonstrated the stability of single-cell proteome profiles in cells under consistent culture conditions.