Xenon Pharmaceuticals Inc., Burnaby, British Columbia, Canada.
Rapid Commun Mass Spectrom. 2024 Feb 15;38(3):e9672. doi: 10.1002/rcm.9672.
Na 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.
Unique peptides for Na 1.1, Na 1.2, and Na 1.6 were selected as quantotropic peptides for each protein and used for their quantification in membranes from stably transfected HEK293 cells and rodent and human brain samples using ultra-high-performance liquid chromatography-electrospray ionization tandem mass spectrometry.
Na 1.1, 1.2, and 1.6 protein expressions in three stably individually transfected HEK293 cell lines were found to be 2.1 ± 0.2, 6.4 ± 1.2, and 4.0 ± 0.6 fmol/μg membrane protein, respectively. In brains, Na 1.2 showed the highest expression, with approximately three times higher (P < 0.003) in rodents than in humans at 3.05 ± 0.57, with 3.35 ± 0.56 in mouse and rat brains and 1.09 ± 0.27 fmol/μg in human brain. Both Na 1.1 and 1.6 expressions were much lower in the brains, with approximately 40% less expression in human Na 1.1 than rodent Na 1.1 at 0.49 ± 0.1 (mouse), 0.43 ± 0.3 (rat), and 0.28 ± 0.04 (humans); whereas Na 1.6 had approximately 60% less expression in humans than rodents at 0.27 ± 0.09 (mouse), 0.26 ± 0.06 (rat), and 0.11 ± 0.02 (humans) fmol/μg membrane proteins.
Multiple reaction monitoring was used to quantify sodium channels Na 1.1, 1.2, and 1.6 expressed in stably transfected HEK293 cells and brain tissues from mice, rats, and humans. We found significant differences in the expression of these channels in mouse, rat, and human brains. Na expression ranking among the three species was Na 1.2 ≫ Na 1.1 > Na 1.6, with the human brain expressing much lower concentrations overall compared to rodent brain.
Na 1.1、1.2 和 1.6 是作为电压门控钠通道发挥作用的跨膜蛋白，与各种形式的癫痫有关。在药物开发过程中，需要了解它们在靶组织中的实际浓度。
为每个蛋白选择了独特的肽作为定量肽，用于通过超高效液相色谱-电喷雾串联质谱法在稳定转染的 HEK293 细胞和啮齿动物和人脑样本的膜中对其进行定量。
在三个稳定单独转染的 HEK293 细胞系中，Na 1.1、1.2 和 1.6 蛋白表达分别为 2.1±0.2、6.4±1.2 和 4.0±0.6 fmol/μg 膜蛋白。在脑中，Na 1.2 表达最高，在啮齿动物中的表达约高 3 倍（P<0.003），在 3.05±0.57 时，在小鼠和大鼠脑中为 3.35±0.56，在人脑为 1.09±0.27 fmol/μg。脑中的 Na 1.1 和 1.6 表达均低得多，人脑 Na 1.1 比啮齿动物 Na 1.1 少约 40%，在 0.49±0.1（小鼠）、0.43±0.3（大鼠）和 0.28±0.04（人）；而 Na 1.6 在人类中的表达比啮齿动物少约 60%，在 0.27±0.09（小鼠）、0.26±0.06（大鼠）和 0.11±0.02（人）中为 0.11±0.02 fmol/μg 膜蛋白。
使用多重反应监测法来定量稳定转染的 HEK293 细胞和来自小鼠、大鼠和人类脑组织中表达的钠通道 Na 1.1、1.2 和 1.6。我们发现这些通道在小鼠、大鼠和人脑中的表达存在显著差异。这三种物种的 Na 表达排名为 Na 1.2≫Na 1.1>Na 1.6，与啮齿动物大脑相比，人类大脑的总体浓度要低得多。
