Superhydrophilic Nanostructured Microparticles for Enhanced Phosphoprotein Enrichment from Alzheimer's Disease Brain

Alzheimer's disease (AD) is an incurable neurodegenerative disorder and closely related to abnormal phosphoproteoforms. The analysis of low-abundance phosphoproteoforms relies heavily on the enrichment of phosphoproteins. However, existing phosphoprotein enrichment materials suffer from either low selectivity or low coverage due to the unavoidable unspecific adsorption of background proteins. Here, we propose a strategy of nanostructure-enabled superhydrophilic surfaces and synthesize Ti⁴⁺-functionalized superhydrophilic nanostructured microparticles (SNMs-Ti⁴⁺) via an emulsion interfacial polymerization process. In this process, hydrophilic and hydrophobic monomers assemble into a stable oil-in-water emulsion, producing microparticles with abundant hydrophilic phosphate nanoprotrusions on the surface. The microparticles are subsequently functionalized with Ti⁴⁺. SNMs-Ti⁴⁺ exhibit enormous nanoprotrusions and abundant Ti⁴⁺ modifications, which allow SNMs-Ti⁴⁺ to effectively adsorb the phosphoproteins and suppress the unspecific adsorption of background proteins. Using these SNMs-Ti⁴⁺, we identified 2256 phosphoproteins from HeLa cells, twice the number of those enriched with commercial kits. From AD mouse brains, 2603 phosphoproteins were successfully enriched, and 10 times of AD-related differentially regulated phosphoproteins were discovered than those without enrichment. These microparticles show great prospects for biomarker detection, disease diagnosis, and downstream biological process disclosure.