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

The human ether-à-go-go-related gene (KCNH2)-encoded protein hERG constitutes the α subunit of the Kv11.1 channel and contributes to the current, which plays an important role in the cardiac action potential. Genetically and xenobiotically triggered malfunctions of hERG can cause arrhythmia. The expression of hERG in various study systems was assessed mainly as the fold change relative to the corresponding control. Here, we developed a simple and sensitive quantitation method using targeted mass spectrometry, i.e., the parallel reaction monitoring approach, to measure the absolute quantity of hERG in copy number. Such measurements do not require controls, and the obtained values can be compared with similar results for any other protein. To effectively avoid matrix effects, we used the heavy-match-light (HML) in-sample calibration approach that requires only a single isotopologue to achieve copy-number quantitation. No significant difference was observed in the results obtained by HML and by the classic standard addition in-sample calibration approach. Using four proteotypic peptides, we quantified the average number of copies of hERG in the HEK293T heterologous expression system as 3.6 ± 0.5 × 10 copies/cell, i.e., 1 million copies/cell for the fully assembled Kv11.1 channel.