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Massively parallel sample preparation for multiplexed single-cell proteomics using nPOP.

Andrew Leduc1, Luke Khoury2, Joshua Cantlon3

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

Nature Protocols
|August 8, 2024
PubMed
Summary
This summary is machine-generated.

We developed nano-proteomic sample preparation (nPOP) for high-throughput single-cell proteomics. This method enables accurate protein quantification in thousands of cells, advancing proteomic analysis scalability.

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Area of Science:

  • Proteomics
  • Mass Spectrometry
  • Cell Biology

Background:

  • Single-cell proteomics using mass spectrometry (MS) offers high specificity and sensitivity.
  • Current methods face limitations in throughput for analyzing large numbers of single cells.

Purpose of the Study:

  • To develop a high-throughput method for single-cell proteomic sample preparation.
  • To enable parallel processing of thousands of single cells for multiplexed MS analysis.

Main Methods:

  • Developed nano-proteomic sample preparation (nPOP) using nanoliter-volume droplets on glass slides.
  • Implemented nPOP with plexDIA (non-isobaric tags) and isobaric tags for multiplexed MS.
  • Utilized a cell-dispensing robot (CellenONE) and readily available consumables.

Main Results:

  • Accurate quantification of ~3,000-3,700 proteins per human cell using plexDIA.
  • Analysis of 1,827 single cells at >1,000 cells/day with 800-1,200 proteins/cell depth using isobaric tags.
  • nPOP protocol takes 1-2 days to prepare >3,000 single cells.

Conclusions:

  • nPOP significantly increases throughput for single-cell proteomics.
  • The protocol is flexible, scalable, and applicable to various MS methods.
  • Quality control software (QuantQC R package) supports robust data analysis and scaling.