Development and application of a multiple reaction monitoring method for the simultaneous quantification of sodium channels Na v 1.1, Na v 1.2, and Na v 1.6 in solubilized membrane proteins from stable HEK293 cell lines, rodents, and human brain tissues

Na 1.1, 1.2, and 1.6 are transmembrane proteins acting as voltage-gated sodium channels implicated in various forms of epilepsy. There is a need for knowing their actual concentration in target tissues during drug development. Unique peptides for Na 1.1, Na 1.2, and Na 1.6 were selected as quantotropic peptides for each protein and used for their quantification in membranes from stably transfected HEK293 cells and rodent and human brain samples using ultra-high-performance liquid chromatography-electrospray ionization tandem mass spectrometry. Na 1.1, 1.2, and 1.6 protein expressions in three stably individually transfected HEK293 cell lines were found to be 2.1 ± 0.2, 6.4 ± 1.2, and 4.0 ± 0.6 fmol/μg membrane protein, respectively. In brains, Na 1.2 showed the highest expression, with approximately three times higher (P Na 1.6, with the human brain expressing much lower concentrations overall compared to rodent brain.