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\begin{document}

\title{Development and Application of an MRM Method for Simultaneous
Quantification of Sodium Channels Na v 1.1, Na v 1.2 and Na v 1.6 in
Stable HEK293 cell lines, and Solubilized Membrane Proteins from Rodents
and Human Brain Tissues.}



\author[1]{Luis Sojo}%
\author[1]{Raibow Kwan}%
\author[1]{Prerna Das}%
\author[1]{Neelan Gerrebos}%
\author[1]{Jenny Li}%
\author[1]{Xin Yin Wang}%
\author[1]{Gina DeBoer}%
\author[1]{Vanessa Emnacen-Pankhurst}%
\author[1]{Sophia Lin}%
\author[1]{Raymond Feng}%
\author[1]{Sam Goodchild}%
\affil[1]{Xenon Pharmaceuticals Inc}%


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  \date{May 23, 2024}


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\begin{abstract}
\textbf{Rationale} Na \textsubscript{v}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.
\textbf{Methods} Unique peptides for Na \textsubscript{v}1.1, Na
\textsubscript{v}1.2 and Na \textsubscript{v}1.6 were selected as
quantotropic peptides for each protein and used for their quantification
in plasma membranes from stably transfected HEK293 cells and rodent and
human brain samples using ultra-high-performance liquid
chromatography/electrospray ionization tandem mass spectrometry.
\textbf{Results} Na \textsubscript{v} 1.1, 1.2 and 1.6 protein
expressions in three stably individually transfected HEK293 cell lines
were found to be 2.1\selectlanguage{ngerman}±0.2, 6.4±1.2 and 4.0±0.6 fmole/\selectlanguage{greek}µ\selectlanguage{english}g membrane protein
respectively. Na \textsubscript{v}1.2 showed the highest expression,
with approximately 3 times higher (p\textless{}0.003) in rodents than in
human at 3.05\selectlanguage{ngerman}±0.57, 3.35±0.56 in mouse and rat brains and 1.09±0.27
fmole/\selectlanguage{greek}µ\selectlanguage{english}g in human, respectively. Both Na \textsubscript{v}1.1 and 1.6
expressions were much lower than Na \textsubscript{v}1.2, with
approximately 40\% less expression in human Na \textsubscript{v}1.1 when
compared with rodents Na \textsubscript{v}1.1 at 0.49\selectlanguage{ngerman}±0.1 (mouse),
0.43±0.3 (rat), and 0.28±0.04 (human); while Na \textsubscript{v}1.6 was
approximately 60\% less expression in human when compared to rodents at
0.27±0.09 (mouse), 0.26±0.06 (rat) and 0.11±0.02 (human) fmole/\selectlanguage{greek}µ\selectlanguage{english}g
membrane proteins. \textbf{Conclusions} MRM was used to quantify sodium
channels Na \textsubscript{v}1.1, 1.2 and 1.6 expressed in stably
transfected HEK293 cells and brain tissues from mouse, rat, and human.
We found significant differences in the expression of these channels in
mouse, rat, and human brains. Na \textsubscript{v} expression ranking
among the three species was Na \textsubscript{v}1.2
\textgreater{}\textgreater{} Na \textsubscript{v}1.1\textgreater{} Na
\textsubscript{v}1.6, with the human brain expressing much lower
concentrations overall in comparison to rodents.%
\end{abstract}%



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\textbf{Hosted file}

\verb`Nav_quantification_Kwan et al_2023_RCMS_Submission_1.docx`  available at \url{https://authorea.com/users/634613/articles/652414-development-and-application-of-an-mrm-method-for-simultaneous-quantification-of-sodium-channels-na-v-1-1-na-v-1-2-and-na-v-1-6-in-stable-hek293-cell-lines-and-solubilized-membrane-proteins-from-rodents-and-human-brain-tissues}

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