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Sensitive profiling of cell surface proteome by using an optimized biotinylation method.

Yanan Li1, Yan Wang1, Jiawei Mao1

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Area of Science:

  • Proteomics
  • Cell Biology
  • Biochemistry

Background:

  • Cell surface proteins are crucial for cellular functions and are accessible targets for therapies and diagnostics.
  • Current cell surface biotinylation methods lack the sensitivity needed for comprehensive proteome profiling.
  • Efficient profiling of cell surface proteins is essential for advancing immunotherapy and biomarker discovery.

Purpose of the Study:

  • To develop an optimized protocol for highly sensitive cell surface proteome profiling.
  • To improve the identification of cell surface proteins from limited cell samples.
  • To enable differential analysis of cell surface proteomes for identifying biomarkers and therapeutic targets.

Main Methods:

  • An optimized cell surface biotinylation protocol was developed and validated.
  • A tip-based fractionation scheme was integrated to enhance protein identification.
  • A pipet tip-based protocol was engineered for processing significantly fewer cells.
  • Label-free quantitative mass spectrometry was employed for differential proteome analysis.

Main Results:

  • The optimized protocol identified 4510 proteins, including 2055 cell surface-associated proteins, from a small protein digest (20 µg).
  • A pipet tip-based protocol enabled the identification of approximately 600 cell surface-associated proteins from 10 times fewer cells.
  • Differential expression of cell surface-associated proteins was observed between BT474 and MCF7 breast cancer cell lines.
  • The new protocols demonstrated high sensitivity, selectivity, ease of use, and time efficiency.

Conclusions:

  • The developed protocols significantly enhance the sensitivity and efficiency of cell surface proteome profiling.
  • These methods allow for in-depth analysis of cell surface proteins from limited cell numbers.
  • The optimized protocols are valuable tools for discovering diagnostic markers and therapeutic targets, with broad applications in cell surface protein studies.