Selective Enrichment and Direct Analysis of Protein S‑Palmitoylation Sites

S-Fatty-acylation is the covalent
attachment of long chain fatty
acids, predominately palmitate (C16:0, S-palmitoylation), to cysteine
(Cys) residues via a thioester linkage on proteins. This post-translational
and reversible lipid modification regulates protein function and localization
in eukaryotes and is important in mammalian physiology and human diseases.
While chemical labeling methods have improved the detection and enrichment
of S-fatty-acylated proteins, mapping sites of modification and characterizing
the endogenously attached fatty acids are still challenging. Here,
we describe the integration and optimization of fatty acid chemical
reporter labeling with hydroxylamine-mediated enrichment of S-fatty-acylated
proteins and direct tagging of modified Cys residues to selectively
map lipid modification sites. This afforded improved enrichment and
direct identification of many protein S-fatty-acylation sites compared
to previously described methods. Notably, we directly identified the
S-fatty-acylation sites of IFITM3, an important interferon-stimulated
inhibitor of virus entry, and we further demonstrated that the highly
conserved Cys residues are primarily modified by palmitic acid. The
methods described here should facilitate the direct analysis of protein
S-fatty-acylation sites and their endogenously attached fatty acids
in diverse cell types and activation states important for mammalian
physiology and diseases.