Premium
On the feasibility of quantifying sodium channel Na v 1.6 protein in mouse brain using targeted ultra‐high‐performance/electrospray ionization multiple reaction monitoring mass spectrometry
Author(s) -
Sojo Luis E.,
Kwan Rainbow,
Dang Cathy,
Tung Matthew,
Li Jenny
Publication year - 2019
Publication title -
rapid communications in mass spectrometry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.528
H-Index - 136
eISSN - 1097-0231
pISSN - 0951-4198
DOI - 10.1002/rcm.8398
Subject(s) - chemistry , electrospray ionization , tandem mass spectrometry , sodium channel , mass spectrometry , in silico , mutant , selected reaction monitoring , trypsin , microbiology and biotechnology , chromatography , biochemistry , enzyme , sodium , biology , organic chemistry , gene
Rationale Na v 1.6 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 Na v 1.6 expression in animal models. This research explores the feasibility of quantifying Na v 1.6 expression in mouse brains using targeted multiple reaction monitoring (MRM) mass spectrometry. Methods A combination of in silico tryptic Na v 1.6 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 Na v 1.6 in induced HEK293 cells. The unique Na v 1.6 peptide, DSLFIPR, was selected as probe for quantifying Na v 1.6 levels in brains from C57BL/6J wild‐type mice as well as two kinds of mutants including Scn8a N1768D/+ and heterozygous null Scn8a +/− mice using isotope dilution targeted mass spectrometry. Results The feasibility of using targeted MRM for quantifying Na v 1.6 expression in mice brains was demonstrated. Expression of Na v 1.6 in brains (hippocampi) from wild‐type and mutant Scn8a N1768D/+ 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. Na v 1.6‐overexpressed HEK293 cells showed 3.7 fmol/μg of Na v 1.6 expression, suitable for screening new compounds for Na v 1.6 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.