Analysis of Protein Cysteine Acylation Using a Modified Suspension Trap (Acyl-Trap)

Proteins
undergo reversible S-acylation via a
thioester linkage in vivo. S-palmitoylation, modification
by C16:0 fatty acid, is a common S-acylation that
mediates critical protein–membrane and protein–protein
interactions. The most widely used S-acylation assays,
including acyl-biotin exchange and acyl resin-assisted capture, utilize
blocking of free Cys thiols, hydroxylamine-dependent cleavage of the
thioester and subsequent labeling of nascent thiol. These assays generally
require >500 μg of protein input material per sample and
numerous
reagent removal and washing steps, making them laborious and ill-suited
for high throughput and low input applications. To overcome these
limitations, we devised “Acyl-Trap”, a suspension trap-based
assay that utilizes a thiol-reactive quartz to enable buffer exchange
and hydroxylamine-mediated S-acyl enrichment. We
show that the method is compatible with protein-level detection of S-acylated proteins (e.g., H-Ras) as well as S-acyl site identification and quantification using “on trap”
isobaric labeling and LC-MS/MS from as little as 20 μg of protein
input. In mouse brain, Acyl-Trap identified 279 reported sites of S-acylation and 1298 previously unreported putative sites.
Also described are conditions for long-term hydroxylamine storage,
which streamline the assay. More generally, Acyl-Trap serves as a
proof-of-concept for PTM-tailored suspension traps suitable for both
traditional protein detection and chemoproteomic workflows.

蛋白质在体内可通过硫酯键发生可逆的S-酰化（S-acylation）修饰。S-棕榈酰化（S-palmitoylation）即由C16:0脂肪酸介导的修饰，是一类常见的S-酰化修饰，可调控关键的蛋白质-膜及蛋白质-蛋白质相互作用。目前应用最广泛的S-酰化检测方法包括酰基-生物素交换法（acyl-biotin exchange）与酰基树脂辅助捕获法（acyl resin-assisted capture），此类方法通过封闭游离半胱氨酸巯基、依赖羟胺的硫酯裂解以及后续新生巯基标记来实现检测。这类检测通常每份样品需要投入超过500微克蛋白质，且需多步试剂移除与洗涤操作，因此操作繁琐，并不适用于高通量与低起始量应用场景。为克服上述局限，本研究开发了“Acyl-Trap”检测体系——一种基于悬浮捕获柱的检测方法，利用巯基反应性石英介质完成缓冲液置换，并通过羟胺介导实现S-酰化蛋白质富集。实验结果表明，该方法可兼容S-酰化蛋白质的蛋白质水平检测（例如H-Ras），同时可通过“柱上”同重同位素标记（isobaric labeling）结合液相色谱-串联质谱（LC-MS/MS）实现S-酰化位点的鉴定与定量，且仅需低至20微克的蛋白质起始量。在小鼠脑组织中，Acyl-Trap成功鉴定到279个已报道的S-酰化位点，以及1298个此前未被报道的潜在位点。本研究同时报道了可实现羟胺长期储存的实验条件，进一步简化了该检测流程。从更广泛的意义上来说，Acyl-Trap为适配传统蛋白质检测与化学蛋白质组学工作流程的、针对翻译后修饰（PTM, post-translational modification）定制的悬浮捕获柱体系提供了概念验证。