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

Abstract

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.

Nanoproteomics enables proteoform-resolved analysis of low-abundance proteins in human serum

“…1a and Supplementary Figs. 2 – 6 ), hereinafter referred to as BAPTES, which we use to silanize the oleic acid coated Fe 3 O 4 NPs 34 following a method for reproducible surface silanization 35 . The terminal allene carboxamide functional group of the BAPTES ligand possesses high chemoselectivity toward the thiol side chain of Cys, and forms a stable and irreversible conjugate not prone to hydrolysis 36 .…”

“…17 ). These results highlight the unique advantages that these functionalized NPs possess for protein enrichment over antibody-based platforms: (a) comparable size and diffusion kinetics to proteins, promoting effective capture of low-abundance proteins within complex mixtures 20 , 47 , 48 ; (b) high surface area per volume and high number of flexible binding sites, contributing to high ligand density and binding efficacy 49 , 50 ; (c) inexpensive, simple, fast, and scalable synthesis with minimal batch-to-batch variability 35 , in contrast to antibodies 14 , 15 , 51 – 53 .…”

Novel Strategies to Address the Challenges in Top-Down Proteomics

“…104 Roberts et al have further developed a reproducible large-scale synthesis of surface silanized Fe 3 O 4 NPs as an enabling nanoproteomics platform and demonstrated its applications for the highly specific enrichment of the human heart phosphoproteome. 103 In this work, a reproducible and largescale (200 mg) synthesis of surface-silanized magnetite (Fe 3 O 4 ) NPs for biological applications was developed. As an alternative to antibodies, these NPs were surface functionalized with a dinuclear Zn(II)-dipicolylamine (Zn-DPA) complex as a phosphoprotein affinity ligand to specifically enrich intact phosphoproteins.…”

MASH Explorer: A Universal Software Environment for Top-Down Proteomics

“…As the popularity of top-down MS-based proteomics grows, MASH software is increasingly becoming a vital and integral tool for users to process complex highresolution top-down LC-MS/MS data. In addition to the case studies of protein identification from human colorectal cancer cell protein extracts 20 and surfactant-extracted protein mixture, 4 as well as the characterization of ADC, 24 many other groups have used the MASH software packages in topdown proteomics projects including analysis of the light and heavy chain connectivity of a monoclonal antibody, 29 characterization of branched ubiquitin chains, 30,31 identification and characterization of intact phosphoproteins, 32 and localization of phosphorylation sites of a phosphatase. 33 As the burgeoning top-down proteomics community continues its rapid growth and has gained momentum through the creation of the Consortium for Top-Down Proteomics (CTDP) (http://www.topdownproteomics.org/), the need for…”