Reproducible large-scale synthesis of surface silanized nanoparticles as an enabling nanoproteomics platform: Enrichment of the human heart phosphoproteome

A reproducible synthetic strategy was developed for facile large-scale (200 mg) synthesis of surface silanized magnetite (Fe3O4) nanoparticles (NPs) for biological applications.After further coupling a phosphate-specific affinity ligand,these functionalized magnetic NPs were used for the highly specific enrichment of phosphoproteins from a complex biological mixture.Moreover,correlating the surface silane density of the silanized magnetite NPs to their resultant enrichment performance established a simple and reliable quality assurance control to ensure reproducible synthesis of these NPs routinely in large scale and optimal phosphoprotein enrichment performance from batch-to-batch.Furthermore,by successful exploitation of a top-down phosphoproteomics strategy that integrates this high throughput nanoproteomics platform with online liquid chromatography (LC) and tandem mass spectrometry (MS/MS),we were able to specifically enrich,identify,and characterize endogenous phosphoproteins from highly complex human cardiac tissue homogenate.This nanoproteomics platform possesses a unique combination of scalability,specificity,reproducibility,and efficiency for the capture and enrichment of low abundance proteins in general,thereby enabling downstream proteomics applications.