Ultradeep Palmitoylomics Enabled by Dithiodipyridine-Functionalized Magnetic Nanoparticles

Abstract

Palmitoylation, a type of fatty acylation, has vital roles in many biological processes. For ultra-deep identification of protein palmitoylation, an enrichment approach based on a novel magnetic microsphere modified with 2,2'-dithiodipyridine (Fe3O4/SiO2-SSPy microsphere) was presented in this study. The Fe3O4/SiO2-SSPy microspheres were synthesized by directly coating thiol-containing silane coupling agent onto the magnetic supraparticles in aqueous solution at room temperature. Due to the intrinsic magnetic properties, high surface-to-volume ratios and abundant reactive functional groups on surface, these microspheres enabled direct capture of palmitoylated targets and convenient isolation, contributing to remarkable enrichment selectivity (purifying palmitoylated peptides from mixtures with non-palmitoylated peptides even at a 1:500 molar ratio) and sensitivity (the detection limit was at fmol level), thus enables a global annotation of protein palmitoylation for complex biological samples. We had successfully identified 1304 putative palmitoylated proteins from mouse brain tissues by using this method, which is the largest mouse palmitoylome dataset to date. Except for those known members, many new proteins and pathways were also found to be regulated by palmitoylation.