Cheung See Kit Melanie, Martin Brent R
Department of Chemistry, Chemical Biology Program, University of Michigan, Ann Arbor, MI, USA.
Methods Mol Biol. 2019;2009:71-79. doi: 10.1007/978-1-4939-9532-5_6.
As the 10-year anniversary of their first introduction approaches, alkynyl fatty acids have revolutionized the analysis of S-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 of S-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 protein S-palmitoylation.
随着炔基脂肪酸首次被引入的10周年纪念日临近，它们彻底改变了S-棕榈酰化动力学的分析方法，作为功能性模拟物被整合到培养细胞的天然修饰位点中。炔基官能团为生物正交的铜（I）催化的叠氮化物-炔烃环加成反应（CuAAC）提供了一个强大的连接点，可与报告基团连接的叠氮化物反应，形成一个稳定的共轭物，用于富集以进行质谱分析或凝胶内荧光分析。重要的是，代谢标记能够对S-棕榈酰化动力学进行时间依赖性分析，这可用于描绘整个蛋白质组中的掺入和周转速率。在这里，我们展示了一种用于细胞标记、点击化学共轭、富集以及用于蛋白质S-棕榈酰化定量质谱分析的等压串联质量标签标记的方案。
