Improved sensitivity in cell surface protein detection by combining chemical labeling with mechanical lysis in a colorectal cancer cell model

Payal Barua, Seong Beom Ahn, Abidali Mohamedali, Fei Liu*

*Corresponding author for this work

    Research output: Contribution to journalArticlepeer-review

    2 Citations (Scopus)

    Abstract

    Objective: To develop a new technique for improved cell surface protein detection and analysis by combining chemical labeling with mild cell lysis using model HCT 116 colorectal cancer cells. 

    Results: We found that Dounce homogenization by hand, rather than the typical sonication or syringe lysis method, recovered surface/membrane proteins more consistently and effectively. This was indicated by marker membrane proteins such as claudin-4 and EGFR (epidermal growth factor receptor) that span the typical 20–200 kD range. As monitored by Western blotting (WB), the Dounce lysis method combined with cell surface biotinylation showed consistent recovery of the marker proteins claudin-4 and EGFR. This lysis method was combined with a cell surface biotinylation strategy to enrich cell surface/membrane proteins using affinity bead-based purification with four-fold less cells compared to prior work. Subsequent LC/MS/MS analysis identified 49 additional surface/membrane proteins for the first time from HCT 116 cells. 

    Conclusion: This combination of methodologies may fit into an advanced workflow for identifying new and elusive cell surface proteins. It can increase the protein coverage for biomarker discovery for colorectal cancer or other cancers. This new detection/analysis approach may also promote new applications in surface display systems as well as cell screening, selection, and binding processes.

    Original languageEnglish
    Pages (from-to)683-695
    Number of pages13
    JournalBiotechnology Letters
    Volume42
    Issue number5
    DOIs
    Publication statusPublished - May 2020

    Keywords

    • Affinity purification
    • Cell surface labeling
    • Colorectal cancer cells
    • Mechanical lysis
    • Proteomics

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