Enrichment of S-Palmitoylated Proteins for Mass Spectrometry Analysis.
Enrichment of S-Palmitoylated Proteins for Mass Spectrometry Analysis.
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用于质谱分析的 S-棕榈酰化蛋白质的富集。
DOI：
10.1007/978-1-4939-9532-5_6
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发表时间：
2019
期刊：
影响因子：
--
通讯作者：
Martin,BrentR
中科院分区：
文献类型：
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作者：
CheungSeeKit,Melanie;Martin,BrentR
随着炔基脂肪酸首次引入10周年的临近，炔基脂肪酸已经彻底改变了s -棕榈酰化动力学的分析，在培养细胞中作为功能模拟物纳入天然修饰位点。炔官能团为Cu(I)催化叠氮化物-炔环加成（CuAAC）到报告器连接的叠氮化物提供了一个强大的生物正交处理，形成一个稳定的共轭物，用于质谱分析或凝胶内荧光富集。重要的是，代谢标记使s -棕榈酰化动力学的时间依赖性分析成为可能，这可以用来描述蛋白质组的结合和周转率。在这里，我们提出了一种用于蛋白质s -棕榈酰化定量质谱分析的细胞标记，点击化学偶联，富集和等压串联质量标签标记的方案。
As the 10-year anniversary of their first introduction approaches, alkynyl fatty acids have revolutionized the analysis ofS-palmitoylation dynamics, acting as functional mimics incorporated into native modification sites in cultured cells. The alkyne functional group provides a robust handle for bioorthogonal Cu(I)-catalyzed azide–alkyne cycloaddition (CuAAC) to reporter-linked azides, forming a stable conjugate for enrichment for mass spectrometry analysis or in-gel fluorescence. Importantly, metabolic labeling enables time-dependent analysis ofS-palmitoylation dynamics, which can be used to profile incorporation and turnover rates across the proteome. Here we present a protocol for cell labeling, click chemistry conjugation, enrichment, and isobaric tandem mass tag labeling for quantitative mass spectrometry analysis of proteinS-palmitoylation.