Antisense oligonucleotides (ASOs) represent a unique category of therapeutics targeting disease-related RNAs. Since this new therapeutic category emerged, the immediate need to analyze ASOs in clinically relevant biological matrices has led to several methodologies, such as ligand binding assays and imaging techniques. To overcome issues in specificity and provide exact quantitative data for ASOs, a new LC-MS/MS method was developed to analyze in brain tissue a novel 4-10-4 gapmer ASO with the potential for treating Parkinson's disease with phosphorothioated backbone and 2'-O-(2-methoxyethyl) modifications. The sample pretreatment protocol to extract the ASO from brain tissue employed solid phase extraction (SPE) and protein digestion. The LC-MS/MS method was fully optimized, validated and applied to quantify the target ASO in brain tissue samples following an in vivo brain distribution study. The method has a Lower Limit Of Quantification of 1 ng/mg and was applied to the study's samples, demonstrating satisfactory sensitivity and providing valuable information about the ASO's distribution in different brain regions over 45 days.

DNA damage-inducible transcript 4 (DDIT4) is a ubiquitous protein whose expression is transiently increased in response to various stressors. Chronic expression has been linked to various pathologies, including neurodegeneration, inflammation, and cancer. DDIT4 is best recognized for repressing mTORC1, an essential protein complex activated by nutrients and hormones. Accordingly, DDIT4 regulates metabolism, oxidative stress, hypoxic survival, and apoptosis. Despite these well-defined biological functions, little is known about its interacting partners and their unique molecular functions. Here, fusing an enhanced ascorbate peroxidase 2 (APEX2) biotin-labeling enzyme to DDIT4 combined with mass spectrometry, the proteins in the immediate vicinity of DDIT4 in either unstressed or acute stress conditions were identified in situ. The context-dependent interacting proteomes were quantitatively but not functionally distinct. DDIT4 had twice the number of interaction partners during acute stress compared to unstressed conditions, and while the two protein lists had minimal overlap in terms of identity, the proteins’ molecular function and classification were essentially identical. Moonlighting keratins and ribosomal proteins dominated the proteomes in both unstressed and stressed conditions, with many of their members having established non-canonical and indispensable roles during stress. Multiple keratins regulate mTORC1 signaling via the recruitment of 14-3-3 proteins, whereas ribosomal proteins control translation, cell cycle progression, DNA repair, and death by sequestering critical proteins. In summary, two potentially distinct mechanisms of DDIT4 molecular function have been identified, paving the way for additional research to confirm and consolidate these findings.