Abstract
Growing clinical evidence reveals that systematic molecular alterations in the brain occur 20 years before the onset of AD pathological features. Apolipoprotein E4 (ApoE4) is one of the most significant genetic risk factors for Alzheimer's disease (AD), which is not only associated with the AD pathological features such as amyloid-β deposition, phosphorylation of tau proteins, and neuroinflammation but is also involved in metabolism, neuron growth, and synaptic plasticity. Multiomics, such as metabolomics and proteomics, are applied widely in identifying key disease-related molecular alterations and disease-progression-related changes. Despite recent advances in the development of analytical technologies, screening the entire profile of metabolites remains challenging due to the numerous classes of compounds with diverse chemical properties that require different extraction processes for mass spectrometry. In this study, we utilized Orbitrap Secondary Ion Mass Spectrometry (OrbiSIMS) as a chemical filtering screening tool to examine molecular alterations in ApoE4-carried neuroglioma cells compared to wild-type H4 cells. The findings were compared using liquid chromatography (LC)-MS/MS targeted metabolomics analysis for the confirmation of specific metabolite classes. Detected alterations in peptide fragments by OrbiSIMS provided preliminary indications of protein changes. These were extensively analyzed through proteomics to explore ApoE4's impact on proteins. Our metabolomics approach, combining OrbiSIMS and LC-MS/MS, revealed disruptions in lipid metabolism, including glycerophospholipids and sphingolipids, as well as amino acid metabolism, encompassing alanine, aspartate, and glutamate metabolism; aminoacyl-tRNA biosynthesis; glutamine metabolism; and taurine and hypotaurine metabolism. Further LC-MS/MS proteomics studies confirmed the dysfunction in amino acid and tRNA aminoacylation metabolic processes, and highlighted RNA splicing alterations influenced by ApoE4.