4.8 Article

Bioorthogonal Conjugation-Assisted Purification Method for Profiling Cell Surface Proteome

期刊

ANALYTICAL CHEMISTRY
卷 94, 期 3, 页码 1901-1909

出版社

AMER CHEMICAL SOC
DOI: 10.1021/acs.analchem.1c05187

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资金

  1. Shenzhen Science and Technology Innovation Commission [JCYJ20180507181659781]
  2. Shenzhen-Hong Kong Science and Technology Innovation Cooperation Zone Shenzhen Park Project [HZQB-KCZYZ-2021017]
  3. The Tung Foundation Biomedical Sciences Centre [9609301, 9609310]
  4. Research Grants Council of Hong Kong [21101917, 11103318, R1020-18F, 11101017]

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Surface biotinylation is widely used to analyze the cellular proteome associated with the plasma membrane. However, it is prone to interference from cytoplasmic biotin-associated proteins. This study established a new workflow (BCAP) that utilizes a bioorthogonal conjugation reaction to minimize interference and efficiently isolate surface proteins.
Surface biotinylation has been widely adapted in profiling the cellular proteome associated with the plasma membrane. However, the workflow is subject to interference from the cytoplasmic biotin-associated proteins that compete for streptavidin-binding during purification. Here we established a bioorthogonal conjugation-assisted purification (BCAP) workflow that utilizes the Staudinger chemoselective ligation to label and isolate surface-associated proteins while minimizing the binding of endogenous biotin-associated proteins. Label-free quantitative proteomics demonstrated that BCAP is efficient in isolating cell surface proteins with excellent reproducibility. Subsequently, we applied BCAP to compare the surface proteome of proliferating and senescent mouse embryonic fibroblasts (MEFs). Among the results, EHD2 was identified and validated as a novel protein that is enhanced at the cell surface of senescent MEFs. We expect that BCAP will have broad applications in profiling cell surface proteomes in the future.

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