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Fine-Tuning of Label-Free Single-Cell Proteomics Workflows.

Pauline Perdu-Alloy1,2, Charline Keller1,2, Anjali Seth3

  • 1Laboratoire de Spectrométrie de Masse BioOrganique (LSMBO), IPHC UMR7178, CNRS, Université de Strasbourg, 25 Rue Becquerel, Strasbourg, Grand Est 67087, France.

Journal of Proteome Research
|March 4, 2026
PubMed
Summary
This summary is machine-generated.

Optimizing single-cell proteomics workflows enhances throughput and protein quantification. This advancement enables robust analysis of cellular heterogeneity using mass spectrometry, paving the way for larger cohort studies.

Keywords:
LC–MS/MS acquisitionSCP sample preparationdia-PASEFdigestion efficiencylabel-free quantificationsingle-cell proteomics (SCP)

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Area of Science:

  • Proteomics
  • Cellular Biology
  • Analytical Chemistry

Background:

  • Single-cell proteomics is crucial for understanding cellular heterogeneity.
  • Current methods face limitations in robustness, reproducibility, and throughput, hindering large-scale studies.
  • Analyzing large single-cell cohorts is essential for statistical confidence.

Purpose of the Study:

  • To optimize mass spectrometry-based single-cell proteomics workflows.
  • To improve robustness, reproducibility, and throughput for analyzing cellular heterogeneity.
  • To establish a reliable method for quantifying thousands of proteins per single cell.

Main Methods:

  • Benchmarking of three nanoElute2-compatible workflows with different sample supports.
  • Comparison of optimized EVO96 workflow with Evosep-based separations at varying throughputs.
  • Evaluation of enzyme/protein ratios for digestion efficiency and chromatographic setup refinement.

Main Results:

  • Established a streamlined, automated sample preparation and direct injection protocol.
  • Achieved quantification of up to 5000 proteins per single HeLa cell.
  • Developed a robust workflow with a throughput of 55 samples per day at 10 min gradient time.

Conclusions:

  • The optimized workflow significantly enhances the capabilities of single-cell proteomics.
  • This advancement addresses key limitations, enabling more in-depth studies of cellular heterogeneity.
  • The method provides a robust and high-throughput solution for large-scale single-cell proteome analysis.