Decoding the Apical-Basal Surfaceome of Colon Epithelial Cells via Side-Selective Biotinylation.

Area of Science:

• Proteomics
• Cancer Biology
• Cell Biology

Background:

• Colorectal cancer (CRC) is a major global health concern.
• Cell surface proteins (CSPs) are crucial for identifying biomarkers and developing therapies for CRC.
• Understanding CSP distribution in polarized CRC cells is limited.

Purpose of the Study:

• To develop a high-throughput method for analyzing CSP distribution in polarized CRC cells.
• To identify side-specific CSPs and potential glycosylation sites.
• To investigate CSP changes in gene-edited CRC models.

Main Methods:

• Developed a method using biotinylation, peptide/protein enrichment, and LC-MS/MS analysis.
• Analyzed polarized CRC epithelial cells (HT29 and HCT116).
• Generated TKS4-knockout cells to assess method sensitivity.

Main Results:

• Simultaneously identified the side-specific distribution of approximately 1200 CSPs.
• Identified nearly 500 potential N-glycosylation sites on these proteins.
• Detected significant differences in surface markers between wild-type and TKS4-knockout cells.

Conclusions:

• The new method provides insights into CSPs in CRC cells and gene-edited models.
• Findings highlight the role of CSPs in TKS4-dependent epithelial-to-mesenchymal transition (EMT)-like phenotypes.
• The study offers potential targets for CRC biomarker and therapeutic development.