Sorek Nadav, Akerman Amir, Yalovsky Shaul
Energy Biosciences Institute, Berkeley, CA, USA.
Methods Mol Biol. 2013;1043:121-34. doi: 10.1007/978-1-62703-532-3_13.
Lipid modifications play a key role in protein targeting and function. The two Arabidopsis Gγ subunits, AGG1 and AGG2, have been shown to undergo prenylation (AGG1) and S-acylation (AGG2). Prenylation involves covalent nonreversible attachment of either farnesyl (15 carbons) or geranylgeranyl (20 carbons) isoprenoids to conserved cysteine residues at or near the C-terminus of proteins. S-acylation, frequently referred to as palmitoylation, involves the attachment of acyl fatty acids to thiol groups of cysteine residues through a reversible thioester bond. The procedures described below allow direct analysis of the prenyl and acyl moieties using gas chromatography-coupled mass spectrometry (GC-MS). These methods are based on (1) cleavage of prenyl groups with the Raney nickel catalyst and (2) analysis of protein S-acylation following cleavage of the acyl fatty acids from proteins by hydrogenation with platinum (IV) oxide. The hydrogenation under these conditions causes an acid transesterification of the acyl moieties, adding an ethyl group to the carboxyl head of the fatty acid. The addition of the ethyl group reduces the polarity of the fatty acids, allowing their efficient separation by gas chromatography.
脂质修饰在蛋白质靶向和功能中起关键作用。拟南芥的两个Gγ亚基AGG1和AGG2已被证明会发生异戊二烯化（AGG1）和S-酰化（AGG2）。异戊二烯化涉及将法尼基（15个碳）或香叶基香叶基（20个碳）类异戊二烯共价不可逆地连接到蛋白质C末端或其附近的保守半胱氨酸残基上。S-酰化，通常称为棕榈酰化，涉及通过可逆硫酯键将酰基脂肪酸连接到半胱氨酸残基的硫醇基团上。以下所述的方法允许使用气相色谱-质谱联用（GC-MS）直接分析异戊二烯基和酰基部分。这些方法基于：（1）用阮内镍催化剂裂解异戊二烯基；（2）在用二氧化铂（IV）氢化从蛋白质上裂解酰基脂肪酸后分析蛋白质的S-酰化。在这些条件下的氢化会导致酰基部分发生酸酯交换，在脂肪酸的羧基头部添加一个乙基。乙基的添加降低了脂肪酸的极性，使其能够通过气相色谱进行有效分离。
