Mass spectrometry analysis of young and aged mice and human Alzheimer's disease with Lewy body pathology using on-slide tissue digestion.

Abstract

Neurodegenerative disorders such as Alzheimer's disease (AD) and Lewy body disease (LBD) are typically diagnosed after irreversible pathology has developed. Aging, the strongest risk factor, drives molecular changes that predispose the brain to synaptic dysfunction and proteinopathy. Glycosylation and extracellular matrix (ECM) remodeling represent underexplored mechanisms linking aging to neurodegeneration, opening avenues for biomarker discovery, yet mass spectrometry-based glycoproteomics and glycomics studies remain limited. Here, we optimized an on-slide tissue digestion workflow for integrated glycome and proteome profiling from 5-mm brain regions from mice and humans using liquid chromatography data-independent acquisition-tandem mass spectrometry (LC-DIA-MS/MS). This workflow was applied to whole brains from age- and sex-stratified wild-type mice (n = 12) and to human postmortem prefrontal cortex tissue from individuals (n = 14) with brainstem- (n = 8) or limbic-predominant (n = 6) LBD, with or without AD co-pathology. DIA substantially increased protein, glycosylated protein, and ECM coverage by two- to threefold relative to traditional data-dependent acquisition (DDA), while library-free DIA searches further improved detection of low-abundance or region-specific proteins. In aged mouse brains, we observed increased levels of synapse-related proteins-including SYNPR, ZNT3, and HPCA-and enrichment of glutamatergic and postsynaptic pathways, reflecting age-associated synaptic remodeling. Glycomics revealed subtle shifts in the sulfation of chondroitin sulfate (CS) disaccharides with age. In human samples, AD-LBD brainstem tissue exhibited significantly reduced unsulfated, 4-O-sulfated, and total CS levels, along with differential expression of ECM, glycosylated, and mitochondrial proteins, and enrichment of mitochondrial pathways compared with LBD brainstem and AD-LBD limbic tissues. These findings indicate that AD co-pathology exerts a region-specific influence on the proteomic and glycomic landscape of LBD. Collectively, this study establishes a robust DIA-based on-slide digestion platform for high-resolution spatial glycomics and proteomics from minimal tissue, revealing aging- and pathology-specific molecular alterations relevant to neurodegeneration and providing a framework for biomarker discovery.