Aim 1): To quantify A) total SCN2A protein and B) mutant SCN2A protein using selected reaction monitoring (SRM) in 3 heterozygous mouse models of SCN2A encephalopathy, each representing a unique clinical phenotype seen in the SCN2A disease population.
Aim 2): To quantify the change in total SCN2A protein, mutant SCN2A protein, and several other proteins in response to an antisense oligonucleotide (ASO) treatment in a gain-of-function (GoF) mouse model of SCN2A encephalopathy using SRM.
BACKGROUND: SCN2A encodes a voltage-gated sodium channel and de novo variants are the cause of autism and a spectrum of developmental and epileptic encephalopathies (DEEs). There is growing evidence to support that the different phenotypes correlate with the resulting functional consequence of the mutations, i.e. whether the protein is loss or gain-of-function.
DESIGN: Brain samples were collected from WT mice and mice heterozygous for SCN2A R854Q, S1759R, or R1883Q at postnatal (P) day 14. R1883Q mice were treated with an ASO that down-regulates SCN2A mRNA or a scrambled, non-specific ASO. Samples were digested with trypsin and analysed on the Agilent 6495 triple quadrupole (QQQ) mass spectrometer coupled to an Agilent 1290 Infinity II liquid chromatography unit.
RESULTS: No significant difference was measured in total SCN2A levels between R1883Q and WT mice injected with the control ASO. R1883Q mice treated with the SCN2A down-regulation ASO had a 3.83 fold reduction in total SCN2A. Tau was significantly increased in R1883Q mice, but was not stabilised to WT levels after ASO treatment.
CONCLUSIONS: Using the Agilent 6495 QQQ instrument we were able to detect protein concentration in a SCN2A disease model and quantify changes in response to ASO treatment. The ability to detect and quantify mutant protein using mass spectrometry shows immense potential as a tool for measuring target engagement for disease-modifying precision medicine treatments.