Relative quantification of membrane associated EGFR from human cancer cell lines via a SILAC based method

Antibody-drug conjugates (ADCs) demonstrate a promising approach in cancer treatment strategies. Generally, ADCs are composed of a monoclonal antibody and a cytotoxic molecule that is conjugated by a linker to the antibody. After specific binding of the monoclonal antibody to the target protein located on the cell surface, the ADC-target protein complex is internalized and releases the drug by proteolytic digestion intracellularly. This causes selective killing of the targeted cancer cell. Therefore, ADCs are specific and toxic at the same time.
Since ADC binding to the target protein is highly dependent on the receptor density on the cell surface, data of receptor densities provide valuable information about the biological background of ADC internalization, ADC efficacy and potency.
To address the evaluation of receptor densities in human cancer, the exemplary target protein EGFR was selected and relatively quantified by applying a SILAC based approach.
Hence, membrane EGFR from four different cell lines were relatively quantified. The cell lines BxPC3, A431, A673 and MDA-MB-468 originated from pancreatic cancer, epidermoid carcinoma, Ewings Sarcoma and breast cancer, respectively. Because previous performed fluorescence activated cell sorting (FACS) investigations have evaluated that the highest EGFR receptor density is present on the MDA-MB-468 cells, this cell line has been defined as reference and was cultivated in heavy medium, which contains isotope labeled lysine and arginine. After sample preparation and subsequent LC-MS/MS analysis, 11 top EGFR peptides were identified and further used for the evaluation process. Relative membrane EGFR quantities were calculated by using the max. intensities of the top 11 EGFR light and heavy peptides. By comparing the EGFR amounts in the membrane fractions of three cell lines with the reference cell line, approximately sixfold more EGFR was detected in A431 cells and twofold more EGFR was present in the BxPC3 cells. No EGFR was discovered in the membrane fraction of the A673 cells. It has been demonstrated that the SILAC based method is suitable for accurate and precise quantification of EGFR located in the membrane fraction of four human cancer cell lines.