Development of an Absolute Quantification Method for hERG Using PRM with Single Isotopologue in-Sample Calibration

Area of Science:

• Biochemistry
• Molecular Biology
• Cardiology

Background:

• The human ether-à-go-go-related gene (hERG) protein forms the Kv11.1 channel crucial for cardiac action potential.
• hERG dysfunction is linked to cardiac arrhythmias.
• Current methods assess hERG expression as fold change, lacking absolute quantification.

Purpose of the Study:

• To develop a simple, sensitive method for absolute hERG protein quantitation.
• To measure hERG in copy number using targeted mass spectrometry.
• To establish a universally comparable protein quantification standard.

Main Methods:

• Utilized parallel reaction monitoring (PRM), a targeted mass spectrometry approach.
• Employed the heavy-match-light (HML) in-sample calibration for matrix effect mitigation.
• Validated HML against the standard addition method.

Main Results:

• Quantified hERG in copy number, independent of controls.
• Achieved precise hERG measurement using four proteotypic peptides.
• Determined average hERG copies in HEK293T cells as 3.6 ± 0.5 × 10^6 copies/cell.

Conclusions:

• The developed PRM method enables accurate absolute quantification of hERG protein.
• This approach facilitates direct comparison of hERG levels across different studies and proteins.
• Provides a robust tool for cardiac safety assessment and drug development.