Ultradeep Palmitoylomics Enabled by Dithiodipyridine-Functionalized Magnetic Nanoparticles

Palmitoylation,
a type of fatty acylation, has vital roles in many
biological processes. For ultradeep identification of protein palmitoylation,
an enrichment approach based on a novel magnetic microsphere modified
with 2,2′-dithiodipyridine (Fe3O4/SiO2-SSPy microsphere) is 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 the surface, these microspheres
enabled direct capture of palmitoylated targets and convenient isolation,
contributing to remarkable enrichment selectivity (purifying palmitoylated
peptides from mixtures with nonpalmitoylated peptides even at a 1:500
molar ratio) and sensitivity (the detection limit was at femtomole
level), thus enabling a global annotation of protein palmitoylation
for complex biological samples. We successfully identified 1304 putative
palmitoylated proteins from mouse brain tissues by using this method,
which is the largest mouse palmitoylome data set to date. Except for
those known members, many new proteins and pathways were also found
to be regulated by palmitoylation.