Comparison of LC-MS/MS-based targeted proteomics and conventional analytical methods for monitoring breast cancer...

Area of Science:

• Biochemistry
• Molecular Biology
• Pharmacology

Background:

• Multidrug resistance (MDR) is a significant challenge in chemotherapy, often driven by ATP-binding cassette (ABC) transporters.
• Breast cancer resistance protein (BCRP) is a key ABC transporter implicated in MDR.
• Existing methods for BCRP quantification are often qualitative or semi-quantitative, leading to incomparable and contradictory results, especially at low resistance levels relevant to clinical scenarios.

Purpose of the Study:

• To develop and validate a precise quantitative method for measuring BCRP expression.
• To accurately assess BCRP levels in cells with low-level drug resistance.
• To provide a reliable tool for understanding the relationship between BCRP and MDR.

Main Methods:

• Established drug-resistant cell lines (SMMC-7721, MCF-7, HepG-2) using mitoxantrone, doxorubicin, and methotrexate.
• Developed and validated a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method using a surrogate peptide for BCRP quantification.
• Compared LC-MS/MS with real-time-polymerase chain reaction (RT-PCR) and Western Blot (WB) for BCRP analysis.

Main Results:

• The developed LC-MS/MS method demonstrated superior sensitivity and stability compared to existing techniques.
• The LC-MS/MS method accurately monitored subtle variations in BCRP expression.
• RT-PCR and WB were found to be less capable of detecting these minor changes in BCRP levels.

Conclusions:

• The LC-MS/MS method offers a robust platform for accurate BCRP quantification, crucial for understanding drug resistance.
• This quantitative approach can help resolve discrepancies in previous BCRP and MDR studies.
• The method is particularly valuable for investigating low-level drug resistance, which is more representative of clinical conditions but understudied.