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Single-Cell Secretome Profiling Enabled by Selective Enrichment and NanoLC-MS/MS Analysis.

Yingyun He1,2, Xinxin Liu1, Fengjiao Zhu3,2

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Angewandte Chemie (International Ed. in English)
|November 28, 2024
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Summary

Researchers developed a novel single-cell secretome profiling method. This technique enhances the detection of low-abundance proteins, offering deeper insights into cellular communication and secretion heterogeneity.

Keywords:
LC–MSalkyne-functionalized micro-reactorclick chemistrysecretome profilingsingle-cell

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Area of Science:

  • Proteomics
  • Cell Biology
  • Biochemistry

Background:

  • Single-cell proteomics allows profiling thousands of proteins from single cells.
  • Investigating the single-cell secretome is challenging due to low-abundance proteins and high-abundance background proteins in serum-containing media.
  • Existing methods struggle with comprehensive secretome analysis at the single-cell level.

Purpose of the Study:

  • To develop a novel method for comprehensive single-cell secretome profiling (SCSP).
  • To overcome the limitations of detecting low-abundance secreted and extracellular vesicle proteins.
  • To enable deeper insights into secretion heterogeneity and intercellular communication.

Main Methods:

  • Integration of metabolic labeling of newly synthesized proteins.
  • Utilized click chemistry-based enrichment for protein isolation.
  • Employed in situ digestion within an alkyne-functionalized capillary micro-reactor.
  • Followed by nano liquid chromatography-tandem mass spectrometry (nanoLC-MS/MS) analysis.

Main Results:

  • Quantified an average of 389 protein groups from the secretome of single HeLa cells (n=17).
  • Confidently identified a total of 752 protein groups in the single-cell secretome.
  • Achieved a significant increase in identified proteins compared to previous antibody-based targeted analyses.

Conclusions:

  • The developed SCSP method provides a powerful tool for single-cell secretome analysis.
  • Enables comprehensive investigation of secretion heterogeneity.
  • Facilitates a deeper understanding of intercellular communication at the single-cell level.