On the feasibility of quantifying sodium channel Na_v1.6 protein in mouse brain using targeted ultra‐high‐performance/electrospray ionization multiple reaction monitoring mass spectrometry

Abstract

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.

Development and application of a multiple reaction monitoring method for the simultaneous quantification of sodium channels Nav1.1, Nav1.2, and Nav1.6 in solubilized membrane proteins from stable HEK293 cell lines, rodents, and human brain tissues

“…Therefore, time courses following the signals of the Na v selected peptides in the presence of Λ‐phosphatase added after the proteolysis of solubilized mouse, rat, and human brain membrane proteins were carried out. In a previous publication, 12 we demonstrated that the peptide DSLFIPIR, used in the quantification of Na v 1.6, did not show signs of phosphorylation. Figure 3A shows the time course of the dephosphorylation experiment using a phosphorylated peptide GAQA S PPPIAPYP S PTR in the presence of Λ‐phosphatase as positive control, where the underscored and bold serine residues indicate the location of phosphorylation, while the same dephosphorylation treatment was done for the selected Na v 1.1, 1.2, and 1.6 peptides in the mouse, rat, and human brain membrane protein samples, is shown in Figure 3B.…”

“…30,31 The mean number of Na v channels in the membrane can be estimated by dividing the mean current per cell by the single channel conductance. We have previously shown that the proposed targeted MRM method differentiates between the Na v 1.6 expression in Scn8a +/À mutant mouse hippocampal tissues (0.2 fmol/μg membrane protein) and their corresponding wild type (0.4 fmol/μg membrane protein), 12 as expected due to the introduced mutation. Similarly, the MRM method was applied to Scn1a +/À mutant mouse hippocampal tissues, which are expected to show about 50% Na v 1.1 expression of the wild type.…”

“…We were able to quantify the expression level of Na v 1.6 in the stably transfected HEK293 cells used in Na v 1.6 modulator potency assessment in vitro , in the mouse whole brains and hippocampal tissues from wild‐type Scn8a and heterozygous null Scn8a +/− mice. The method showed a 50% reduction in the expression of Na v 1.6 in the heterozygous null Scn8a +/− mice compared to their wild‐type phenotypes as expected 12 . More recently, a similar approach was used to quantify transiently expressed Na v 1.5 channels in Chinese hamster ovary cells 14 .…”

“…The method showed a 50% reduction in the expression of Na v 1.6 in the heterozygous null Scn8a +/À mice compared to their wild-type phenotypes as expected. 12 More recently, a similar approach was used to quantify transiently expressed Na v 1.5 channels in Chinese hamster ovary cells. 14 The method described in this work is an extension of our previous work to include Na v 1.1 and 1.2 sodium channels, and therefore, it is still a feasibility study to demonstrate its potential in quantifying these proteins in the stably transfected HEK293 cell lines as well as in mouse, rat and human brain tissues in support of animal studies in epilepsy.…”

“…Mass spectrometry (MS) continues to be a key enabling technology in the study of biological systems and certainly the main drivers in bioanalytical chemistry. [8][9][10][11] In our recent publication, 12 we demonstrated for the first time the use of targeted MS using multiple reaction monitoring (MRM) for quantifying Na v 1.6 in transfected HEK293 cells, whole brains, and hippocampal tissues from laboratory mice. This was achieved by a combination of in silico peptide MRM predictions using Skyline, 13 a standard proteomic workup including membrane protein isolation, trypsin-based proteolysis, and ultrahigh-pressure liquid chromatography coupled to electrospray mass spectrometry (UHPLC/ESI-MS/MS).…”