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.

S-脂肪酰化（S-fatty-acylation）指长链脂肪酸（主要为棕榈酸C16:0，即S-棕榈酰化（S-palmitoylation））通过硫酯键与蛋白质的半胱氨酸（Cys）残基发生共价结合的修饰过程。该修饰属于翻译后可逆的脂质修饰，可调控真核生物体内蛋白质的功能与定位，在哺乳动物生理活动及人类疾病进程中具有重要意义。尽管化学标记方法已提升了S-脂肪酰化蛋白质的检测与富集效率，但精准定位修饰位点并表征内源性结合的脂肪酸仍存在较大挑战。本研究将脂肪酸化学报告基团标记技术与羟胺介导的S-脂肪酰化蛋白质富集策略、修饰半胱氨酸残基的直接标记技术相结合并进行优化，以选择性绘制脂质修饰位点图谱。相较于此前报道的方法，该策略可实现更优异的富集效果，并可直接鉴定出大量蛋白质的S-脂肪酰化位点。值得注意的是，我们直接鉴定了IFITM3的S-脂肪酰化位点——该蛋白是重要的病毒入胞干扰素诱导抑制剂——并进一步证实其高度保守的半胱氨酸残基主要被棕榈酸修饰。本文所述方法将有助于在与哺乳动物生理及疾病相关的多种细胞类型与激活状态下，直接分析蛋白质的S-脂肪酰化位点及其内源性结合的脂肪酸。