Comparison of liquid chromatography–tandem mass spectrometry-based targeted proteomics and conventional analytical methods for the determination of P-glycoprotein in human breast cancer cells

• A novel and advanced LC/MS/MS-based targeted proteomics assay was developed and validated.
• Protein digestion efficiency and membrane extraction recovery were discussed.
• The LC/MS/MS assay was compared with the conventional analytical methods.
• The LC/MS/MS assay can allow the measurement of the P-gp expression level in a more accurate manner.

P-glycoprotein (P-gp) is the most frequently proposed factor for multi-drug resistance. It is traditionally measured using antibody-based methods. While these techniques can provide relative quantification values for P-gp levels, the important information that is usually missing is its amount in the biological system. In this study, a novel and advanced liquid chromatography–tandem mass spectrometry (LC/MS/MS)-based targeted proteomics assay was developed and validated for the determination of P-gp in the breast cancer drug sensitive cell line MCF-7/WT and the drug resistant cell line MCF-7/ADR. Three tryptic peptides (434STTVQLMQR442, 674GSQAQDR680 and 368IIDNKPSIDSYSK380) can specifically represent P-gp. Among these peptides, 434STTVQLMQR442 was selected as the surrogate analyte for quantification, and a stable isotope-labeled synthetic peptide with the same sequence was used as an internal standard. The calibration range was validated from 10 to 1000 ng/mL. The intra- and inter-day precisions were within 5.9% and 3.7%, respectively. The accuracy for the quality control (QC) samples was within 8.0%. Using this assay, the amounts of P-gp were accurately quantified as 3.53 fg/cell (∼2.08 × 10−2 amol/cell) in the MCF-7/WT cells and 34.5 fg/cell (∼2.02 × 10−1 amol/cell) in the MCF-7/ADR cells. This outcome was then compared with those obtained by conventional analytical methods including confocal microscopy, western blotting and flow cytometry. The comparative results show that not only is the LC/MS/MS-based targeted proteomics assay able to monitor the protein levels in a more accurate manner, but the large discrepancy observed between the other methods was most likely due to the lack of specificity and the semi-quantitative nature of the conventional assays