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Related Experiment Video

Updated: Jun 23, 2025

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Efficient and Sensitive Sample Preparation, Separations, and Data Acquisition for Label-Free Single-Cell Proteomics.

Thy Truong1, Ximena Sanchez-Avila1, Kei G I Webber1

  • 1Department of Chemistry and Biochemistry, Brigham Young University, Provo, UT, USA.

Methods in Molecular Biology (Clifton, N.J.)
|June 21, 2024
PubMed
Summary
This summary is machine-generated.

This study introduces a fast, label-free workflow for single-cell proteomics, enabling researchers to quantify thousands of proteins per cell. The method enhances accessibility to proteome profiling for more laboratories.

Keywords:
HP D100 single cell dispenserLabel-free quantificationNanoLCOne-step workflowSingle-cell proteomicsWide window acquisition

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Area of Science:

  • Proteomics
  • Mass Spectrometry
  • Cell Biology

Background:

  • Single-cell proteomics is crucial for understanding cellular heterogeneity.
  • Existing methods often lack sensitivity or require specialized expertise.
  • Label-free approaches offer advantages in workflow simplicity.

Purpose of the Study:

  • To develop a sensitive and efficient label-free workflow for single-cell proteomics.
  • To enable broad accessibility of single-cell proteome profiling.
  • To streamline sample preparation and data acquisition processes.

Main Methods:

  • Utilized the Tecan Uno Single Cell Dispenser for rapid cell isolation and nanoliter reagent dispensing.
  • Developed a streamlined sample processing workflow for cell lysis, denaturation, and digestion within 1 hour.
  • Employed low-flow liquid chromatography coupled with wide-window data-dependent acquisition on an Orbitrap Exploris 480 mass spectrometer.

Main Results:

  • Achieved quantification of nearly 3000 proteins per single cell.
  • Demonstrated a sensitive and efficient workflow for label-free single-cell proteomics.
  • The workflow integrates sample preparation, liquid chromatography, and mass spectrometry.

Conclusions:

  • The developed workflow significantly enhances accessibility to sensitive single-cell proteome profiling.
  • This method is suitable for nonspecialist laboratories, broadening research capabilities.
  • The approach provides a robust platform for deep proteome analysis at the single-cell level.