Thinon Emmanuelle, Hang Howard C
Laboratory of Chemical Biology and Microbial Pathogenesis, The Rockefeller University, New York, NY, USA.
Molecular Cell Biology of Autophagy, The Francis Crick Institute, London, UK.
Methods Mol Biol. 2019;2009:45-57. doi: 10.1007/978-1-4939-9532-5_4.
Protein S-fatty-acylation, the covalent addition of a long-chain fatty acid, predominantly palmitate (S-palmitoylation), to cysteine, is a highly dynamic and regulated process that controls protein function and localization of membrane-associated proteins in eukaryotes. The analysis of S-fatty acylated peptides by mass spectrometry remains challenging due to the hydrophobic and potentially labile thioester linkage of the S-fatty acylated peptides.Here we describe an optimized protocol for the global analysis of S-palmitoylated proteins based on the combination of an alkyne-tagged chemical reporter of palmitoylation, alk-16 with hydroxylamine-selective hydrolysis of thioester bonds. This protocol decreased the number of false positive proteins and was applied to identify S-fatty acylation sites, providing modification sites for 44 proteins out of the 106 S-fatty acylated proteins identified.
蛋白质S-脂肪酸酰化是指将长链脂肪酸（主要是棕榈酸，即S-棕榈酰化）共价添加到半胱氨酸上的过程，这是一个高度动态且受调控的过程，可控制真核生物中膜相关蛋白的功能和定位。由于S-脂肪酸酰化肽段具有疏水且潜在不稳定的硫酯键，通过质谱分析S-脂肪酸酰化肽段仍然具有挑战性。在此，我们描述了一种基于炔烃标记的棕榈酰化化学报告分子alk-16与硫酯键的羟胺选择性水解相结合的优化方案，用于全面分析S-棕榈酰化蛋白。该方案减少了假阳性蛋白的数量，并应用于鉴定S-脂肪酸酰化位点，在鉴定出的106个S-脂肪酸酰化蛋白中，为44个蛋白提供了修饰位点。
