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Bioorthogonal Conjugation-Assisted Purification Method for Profiling Cell Surface Proteome.

Guopan Liu1,2, Ming Ho Choi1, Haiying Ma1

  • 1Department of Biomedical Sciences, College of Veterinary Medicine and Life Sciences, City University of Hong Kong, 83 Tat Chee Avenue, Kowloon 999077, Hong Kong China.

Analytical Chemistry
|January 12, 2022
PubMed
Summary
This summary is machine-generated.

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This study introduces a new method, bioorthogonal conjugation-assisted purification (BCAP), to accurately profile cell surface proteins. BCAP effectively isolates surface proteins, revealing new insights into cellular changes during senescence.

Area of Science:

  • Proteomics
  • Cell Biology
  • Biochemistry

Background:

  • Surface biotinylation is common for studying cell surface proteins but is hindered by cytoplasmic protein contamination.
  • Existing methods struggle to differentiate between surface-localized and intracellular biotinylated proteins.

Purpose of the Study:

  • To develop a novel, highly specific method for isolating and profiling cell surface proteins.
  • To overcome the limitations of traditional biotinylation techniques by minimizing non-specific binding.
  • To compare the surface proteomes of proliferating and senescent cells.

Main Methods:

  • Established a bioorthogonal conjugation-assisted purification (BCAP) workflow.
  • Utilized Staudinger chemoselective ligation for specific labeling of surface proteins.

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  • Employed label-free quantitative proteomics for analysis.
  • Applied BCAP to mouse embryonic fibroblasts (MEFs) in different states (proliferating vs. senescent).

    • Main Results:

    • BCAP demonstrated high efficiency and reproducibility in isolating cell surface proteins.
    • The method successfully minimized the interference from endogenous biotinylated cytoplasmic proteins.
    • Identified EHD2 as a novel protein upregulated at the cell surface of senescent MEFs compared to proliferating MEFs.

    Conclusions:

    • BCAP is a robust and specific technique for cell surface proteomics.
    • The findings highlight BCAP's potential for discovering biomarkers and understanding cellular processes.
    • BCAP is expected to be broadly applicable for future cell surface proteome profiling studies.