Sojo Luis E, Kwan Rainbow, Dang Cathy, Tung Matthew, Li Jenny
Compound Properties Group, Xenon Pharmaceuticals Inc, 3650 Gilmore Way, Burnaby, British Columbia, V5G 4W8, Canada.
Department Chemistry, Simon Fraser University, 8888 University Drive, Burnaby, British Columbia, V5A 1S6, Canada.
Rapid Commun Mass Spectrom. 2019 Apr 15;33(7):683-696. doi: 10.1002/rcm.8398.
Na 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 1.6 expression in animal models. This research explores the feasibility of quantifying Na 1.6 expression in mouse brains using targeted multiple reaction monitoring (MRM) mass spectrometry.
A combination of in silico tryptic Na 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 1.6 in induced HEK293 cells. The unique Na 1.6 peptide, DSLFIPR, was selected as probe for quantifying Na 1.6 levels in brains from C57BL/6J wild-type mice as well as two kinds of mutants including Scn8a and heterozygous null Scn8a mice using isotope dilution targeted mass spectrometry.
The feasibility of using targeted MRM for quantifying Na 1.6 expression in mice brains was demonstrated. Expression of Na 1.6 in brains (hippocampi) from wild-type and mutant Scn8a 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 1.6-overexpressed HEK293 cells showed 3.7 fmol/μg of Na 1.6 expression, suitable for screening new compounds for Na 1.6 blocking activity.
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.
钠通道1.6（Na 1.6）是一种跨膜电压门控钠通道，与多种形式的癫痫有关。在癫痫动物模型中，可使用抑制剂调节其活性，这可能导致其表达发生变化。需要在动物模型中对Na 1.6表达进行可靠的定量测量。本研究探讨了使用靶向多反应监测（MRM）质谱法定量小鼠脑中Na 1.6表达的可行性。
结合计算机模拟的胰蛋白酶消化Na 1.6肽段和MRM跃迁来选择目标肽段。随后进行简单的蛋白质组学处理，包括质膜分离、基于胰蛋白酶的蛋白水解以及超高效液相色谱/电喷雾电离串联质谱（UHPLC/ESI-MS/MS），以检测诱导的HEK293细胞中Na 1.6的存在。选择独特的Na 1.6肽段DSLFIPR作为探针，使用同位素稀释靶向质谱法定量C57BL/6J野生型小鼠以及包括Scn8a和杂合缺失Scn8a小鼠在内的两种突变体小鼠脑中的Na 1.6水平。
证明了使用靶向MRM定量小鼠脑中Na 1.6表达的可行性。发现野生型和突变型Scn8a小鼠脑（海马体）中Na 1.6的表达约为0.40 fmol/μg。另一方面，突变型缺失Scn8a杂合小鼠的水平为0.22 fmol/μg，基于这种仅产生野生型小鼠50%表达的特定突变，这一结果符合预期。过表达Na 1.6的HEK293细胞显示Na 1.6表达为3.7 fmol/μg，适用于筛选具有Na 1.6阻断活性的新化合物。
本可行性研究结果支持使用DSLFIPIR通过UHPL/ESI-MS/MS定量脑组织中的Nav1.6。
