On the feasibility of quantifying sodium channel Nav1.6 protein in mouse brain using targeted ultra‐high‐performance/electrospray ionization multiple reaction monitoring mass spectrometry

Rationale Nav1.6 is a transmembrane voltage gated sodium channel implicated in various forms of epilepsy. Modulation of its activity in epilepsy animal models can be accomplished using inhibitors which may result in changes in its expression. There is a need to generate reliable quantitative measurements of Nav1.6 expression in animal models. This research explores the feasibility of quantifying Nav1.6 expression in mouse brains using targeted multiple reaction monitoring (MRM) mass spectrometry. Methods A combination of in silico tryptic Nav1.6 peptides and MRM transitions were used to select target peptides. This was followed by a simple proteomic work‐up including plasma membrane isolation, trypsin‐based proteolysis and ultra‐high‐performance/electrospray ionization tandem mass spectrometry (UHPLC/ESI‐MS/MS) to detect the presence of Nav1.6 in induced HEK293 cells. The unique Nav1.6 peptide, DSLFIPR, was selected as probe for quantifying Nav1.6 levels in brains from C57BL/6J wild‐type mice as well as two kinds of mutants including Scn8aN1768D/+ and heterozygous null Scn8a+/− mice using isotope dilution targeted mass spectrometry. Results The feasibility of using targeted MRM for quantifying Nav1.6 expression in mice brains was demonstrated. Expression of Nav1.6 in brains (hippocampi) from wild‐type and mutant Scn8aN1768D/+ mice were found to be around 0.40 fmol/μg. Mutant null Scn8a+/− heterozygous mice, on the other hand, showed levels of 0.22 fmol/μg as expected based on this particular mutation which only generates 50% of the expression in wild‐type mice. Nav1.6‐overexpressed HEK293 cells showed 3.7 fmol/μg of Nav1.6 expression, suitable for screening new compounds for Nav1.6 blocking activity. Conclusions The results of the present feasibility study support the use of DSLFIPIR for quantification of Nav1.6 in brain tissues using UHPL/ESI‐MS/MS.