Orthogonal Ionic Liquid‐Based Extraction Strategy Enables Amyloid‐Specific Profiling of Aggregate Proteome

ABSTRACT Amyloid deposits, composed of insoluble cross‐β‐sheet fibrils, are pathological hallmarks of many neurodegenerative diseases. However, their structural heterogeneity and extreme insolubility hinder characterization of their molecular composition and deposition mechanisms. Here, an orthogonal ionic liquid‐based extraction strategy (Orth‐ i EA) was developed to selectively enrich amyloid aggregates from complex biological samples. Systematic screening identified tetramethylguanidine tetrafluoroborate (TMGBF ₄ ) as a potent ionic liquid capable of disrupting hydrogen bonds within cross‐β‐sheet structures, thereby solubilizing amyloid aggregates. In contrast, 1‐dodecyl‐3‐methylimidazolium chloride (C12ImCl) preferentially solubilized non‐amyloid proteins through hydrophobic interactions. In combination, these two reagents constitute the orthogonal extraction system, enabling the highly selective enrichment of amyloid aggregates from complex biological samples while remaining compatible with downstream LC‐MS/MS proteomic workflows after mild desalting. Application of Orth‐ i EA to hippocampal tissue from 12‐month‐old 3xTg Alzheimer's disease model mice identified numerous amyloid‐associated proteins enriched in mitochondrial components, which was further confirmed by immunofluorescence co‐localization analysis. Functional and network analyses converged on pathways involving protein transport, mitochondrial translation, intracellular signaling, and apoptosis, revealing previously unrecognized molecular links between amyloid pathology and Alzheimer's disease. Therefore, Orth‐ i EA provides a versatile chemical platform for selective isolation and molecular profiling of amyloid aggregates.