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Related Experiment Video

Updated: Aug 17, 2025

Characterizing Cellular Proteins with In-cell Fast Photochemical Oxidation of Proteins
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Exploring functional protein covariation across single cells using nPOP.

Andrew Leduc1, R Gray Huffman2, Joshua Cantlon3

  • 1Departments of Bioengineering, Biology, Chemistry and Chemical Biology, Single Cell Proteomics Center, and Barnett Institute, Northeastern University, Boston, MA, 02115, USA. leduc.an@northeastern.edu.

Genome Biology
|December 16, 2022
PubMed
Summary

A new method, nPOP, enables high-throughput single-cell proteomics by automating sample preparation for thousands of cells. This reveals cell cycle differences and energy metabolism variations in melanoma subpopulations, advancing our understanding of drug resistance.

Keywords:
Cell division cycleDrug resistanceProtein covariationSample preparationSingle cellSingle-cell proteomics

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Area of Science:

  • Proteomics
  • Cell Biology
  • Biochemistry

Background:

  • Protein covariation across single cells reflects key biological processes like cell division and drug resistance.
  • Single-cell mass spectrometry offers a way to quantify and interpret this protein covariation with high throughput and accuracy.

Purpose of the Study:

  • To introduce nPOP, a novel method for simultaneous, automated sample preparation for thousands of single cells.
  • To utilize nPOP for analyzing protein covariation and understanding cell cycle dynamics and metabolic differences in melanoma cells.

Main Methods:

  • nPOP employs piezo acoustic dispensing for isolating individual cells in picoliter volumes.
  • Simultaneous lysis, digestion, and labeling of thousands of single cells are performed in nanoliter volumes on a specialized slide.
  • Protein covariation analysis is conducted using mass spectrometry.

Main Results:

  • nPOP successfully identified cell cycle dynamics and differences between cell types, including melanoma subpopulations.
  • Melanoma cells with drug resistance markers showed distinct cell cycle progression, protein covariation, glycogen accumulation, and altered enzyme abundance.
  • Analysis revealed inverse covariation between oxidative phosphorylation and glycolysis proteins in non-primed melanoma cells, suggesting divergent energy metabolism.

Conclusions:

  • nPOP provides a flexible, automated, and highly parallelized approach for single-cell proteomics.
  • The method enables the quantification of protein covariation across thousands of cells, revealing functional differences between cell states.
  • nPOP facilitates the discovery of biologically concerted differences in closely related cell states.