Optimization of sample preparation for transporter protein quantification in tissues by LC-MS/MS

Area of Science:

• Pharmacology
• Biochemistry
• Analytical Chemistry

Background:

• Accurate quantification of drug transporter proteins in tissues is vital for predicting drug disposition.
• Existing mass spectrometry methods often yield high variability in transporter protein quantification.
• Optimization of these methods is needed to improve reproducibility and reliability.

Purpose of the Study:

• To evaluate and optimize mass spectrometry-based quantification methods for drug transporter proteins in tissues.
• To improve the precision and yield of transporter protein measurements.
• To reduce variability in the quantification of drug transporter proteins.

Main Methods:

• Compared three plasma membrane (PM) protein isolation protocols: commercial kit, Potter-Elvehjem homogenization with ultracentrifugation, and Tween 40 enrichment.
• Evaluated five different protein digestion protocols, including Lys-C, trypsin, enhancer, and heat denaturation.
• Quantified transporter proteins using liquid chromatography-tandem mass spectrometry in selected reaction monitoring mode.

Main Results:

• The Potter-Elvehjem homogenization/ultracentrifugation and Tween 40 enrichment methods yielded approximately two times higher transporter protein quantity (e.g., Bcrp) compared to the commercial kit.
• The optimal digestion protocol involved Lys-C and trypsin with a trypsin enhancer and heat denaturation.
• The optimized method increased quantified Bcrp and Na+/K+-ATPase levels by two to three times compared to previous reports, with a coefficient of variation below 10%.

Conclusions:

• An improved methodology for transporter protein quantification was established by optimizing PM isolation and protein digestion.
• The optimized procedure significantly enhances transporter protein yield and precision.
• This advancement is critical for reliable drug disposition studies.