PROTEOLIPIDOME ANALYSIS OF HIPPOCAMPAL TISSUE FROM A MURINE ALZHEIMER’S DISEASE MODEL TO FACILITATE THERAPEUTIC TARGET IDENTIFICATION

Abstract

Mass spectrometry-based proteomics has potential for uncovering the molecular mechanisms underlying Alzheimer's Disease (AD). However, proteins with atypical biochemical/biophysical properties may go understudied as they may not survive the typical proteomics workflow. Utilizing a unique approach, we have assessed differences across the proteolipidome between the E4FAD murine model of AD and wildtype mice. A liquid-liquid extraction adapted from the Bligh and Dyer method [J. Biochem. Physiol. 1959] was used to isolate proteolipids from hippocampal tissue from apoliprotein E4-5XFAD transgenic and wild-type C57BL/6 mice. Proteolipids were digested with trypsin, labeled with isobaric tandem mass tags (TMT) to provide relative quantitation. Peptides were injected onto a laser-pulled nanobore C18 column and resolved on a liquid chromatograph using an optimized 3-hour gradient coupled to a hybrid quadrupole-Orbitrap mass spectrometer operating in the dd-MS2 mode. Relative peptide abundance was determined using a Proteome Discoverer 2.2. Proteins with a p-value ≤ 0.05 that exhibited a log2-fold change of 0.5 or greater were subjected to functional protein association network analysis as well as comprehensive gene set enrichment analysis. By comparison with results obtained using an optimized bottom-up proteomics workflow, it is evident that many of the proteins found in this experiment are unique to this technique and do not appear in experiments using standard detergent-based aqueous extraction buffers. The relative abundance of many proteins differed between E4FAD and wildtype hippocampi and Protein Center gene ontology analysis revealed numerous proteins to be membrane-specific with protein binding functions. Additionally, many proteins had post-translational modifications (PTMs) such as phosphorylation and acetylation, revealing an interconnected signaling network of proteins that differ in wildtype and E4FAD hippocampus. This novel approach analyzing organically extracted proteolipid fractions provides supplemental coverage of the proteome not revealed by conventional proteomics workflows, including changes in protein expression and PTMs distinctive for the E4FAD as compared to wildtype mice. This method has provided new information regarding the pathological mechanisms in this model, and will be used to facilitate identification of new targets for potential therapeutic development in the treatment of AD.