SILAC-based quantitative MS approach for real-time recording protein-mediated cell-cell interactions

Area of Science:

• Biochemistry
• Proteomics
• Cancer Biology

Background:

• Tumor microenvironment cell interactions are crucial for cancer progression.
• Analyzing secreted proteins between cancer cells and stroma reveals molecular mechanisms of cell-cell interactions.

Purpose of the Study:

• To develop and validate novel stable isotope labeling of amino acids in cell culture (SILAC)-based mass spectrometry (MS)/MS approaches for quantifying protein secretion changes.
• To compare the efficiency and economy of spike-in SILAC and triple-SILAC methods in co-culture systems.

Main Methods:

• Developed two SILAC-based MS/MS approaches: spike-in SILAC and triple-SILAC.
• Utilized CT26 and Ana-1 cell co-culture system for initial method development and validation.
• Applied methods to human cell lines (NCM460 and HT29) to assess feasibility.

Main Results:

• Both spike-in SILAC and triple-SILAC quantitatively measured protein secretion changes in mouse cell co-cultures.
• Validated quantified proteins (Galectin-1, Cathepsin L1, Thrombospondin-1) using Western blotting, showing good agreement with SILAC results.
• Confirmed the capability of both SILAC methods to monitor altered secreted proteins in human cell line co-cultures.

Conclusions:

• Spike-in SILAC and triple-SILAC are effective for quantifying protein secretion changes in co-cultured cells.
• Triple-SILAC demonstrates superior efficiency and economy for real-time monitoring of secreted proteins in the tumor microenvironment compared to spike-in SILAC.