Quantitative Analysis Of Drug Resistance - Related Membrane Proteins In Breast Cancer By Using Directed Proteomics Liquid Level Tandem Technique

Purpose: The high incidence of multi-drug resistance(MDR) of breast cancer chemotherapy was associated with membrane protein, including P-glycoprotein(P-gp) and transferrin receptor(Tf R) which are distributed in the cell membrane. P-gp can efflux drugs from cancer cells and its overexpression is commonly associated with multi-drug resistance. The accurate quantification of P-gp would help predict the response to chemotherapy and for prognosis of breast cancer patients. Tf R is involved in iron absorption and plays an important role in cell growth and cell proliferation. Overexpression of Tf R has been described qualitatively in various cancers, including breast cancer. The expression of Tf R was more in drug-resistant cancer cells and breast cancer tissue. Quantitative determination of contents of P-gp and Tf R in breast cancer will help improve the early detection of cancer diagnosis and prediction of chemotherapeutic effect and prognosis in patients with breast cancer.Methods: A novel and advanced liquid chromatography–tandem mass spectrometry(LC-MS/MS)-based targeted proteomics assay was developed and validated for monitoring P-gp and Tf R levels in human breast cells and tissue samples.Results: 1. Three tryptic peptides(434STTVQLMQR442, 674GSQAQDR680 and 368IIDNKPSIDSYSK380) can specifically represent P-gp. 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 amounts of P-gp were accurately quantified in the MCF-7/WT cells and MCF-7/ADR cells. This outcome was then compared with those obtained by conventional analytical methods including confocal microscopy, western blotting and flow cytometry. Matched pairs of breast tissue samples from 60 patients who were suspected to have drug resistance were subject to analysis. The levels of P-gp were also quantified.2. The tryptic peptide 681VEYHFLSPYVSPK693 of Tf R was selected as the surrogate peptide for quantification and used a stable isotope-labeled synthetic peptide with this same sequence as an internal standard. Quality control data indicated acceptable accuracy and precision. Finally, this assay was successfully applied to the quantitative analysis of Tf R in three breast cell lines and 36 matched pairs of breast tissue samples. The experimental values were compared with those obtained from conventional analytical methods, including immunofluorescence microscopy, western blotting, flow cytometry, and immunohistochemistry.Conclusion: The results showed that not only the LC-MS/MS-based targeted proteomics assay was a more accurate method for the determination of P-gp and Tf R levels in cells and tissues, but also afford more reliable quantification of proteins at low concentrations in clinical practice. This approach could provide the possibility of large-scale determination of biomarkers in the future.In conclusion, in our study, LC-MS/MS assay showed its many advantages as followings, higher sensitivity, higher efficience, better selectivity, less cost, lower limit of quantification(LOQ) and especially an offer of more reliable data when analyzing samples with lower concentrations and biological samples. The LC-MS/MS was proved to be an effective quantification technology for clinical samples.