Probing lysine acetylation with a modification-specific marker ion using high-performance liquid chromatography/electrospray-mass spectrometry with collision-induced dissociation
Probing lysine acetylation with a modification-specific marker ion using high-performance liquid chromatography/electrospray-mass spectrometry with collision-induced dissociation
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DOI：
10.1021/ac0256080
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发表时间：
2002-11-01
影响因子：
7.4
通讯作者：
Yoo, JS
中科院分区：
文献类型：
--
作者：
Kim, JY;Kim, KW;Yoo, JS
蛋白质的翻译后乙酰化调节许多不同的功能，包括DNA识别、蛋白质-蛋白质相互作用和蛋白质稳定性。对调节蛋白质乙酰化的酶的鉴定揭示了这种修饰比之前怀疑的更广泛的用途。在这项研究中，我们描述了一种通过使用带有新的修饰特异性标记离子的高效液相色谱/电喷雾质谱联用分析消化蛋白质来鉴定赖氨酸残基上的蛋白质乙酰化的方法。用毛细管或纳米LC/ESI-TOF-MS碰撞诱导解离，得到可作为乙酰化赖氨酸标志物的碎片离子。虽然m/z=143.1的乙酰化赖氨酸离子已被用作检测乙酰化赖氨酸的标记离子，但在某些多肽中，它会与内部碎片离子混淆，产生假阳性结果。我们在m/z 126.1处发现了一个新的标记离子，这是在m/z 143.1处乙酰化赖氨酸亚胺离子失去NH_3而引起的进一步碎片离子。在m/z为143.1的情况下，发现标记离子比免疫离子具有更高的特异度和9倍的灵敏度。此外，在m/z为126.1的提取离子色谱图中，未发现乙酰化肽的干扰离子。以乙酰化细胞色素c为模型化合物，验证了该方法的实用性。用新的标记离子探测修饰后，用CID-MS光谱确定了乙酰化赖氨酸的位置。将该方法应用于经曲古抑素A处理的HeLa细胞中组蛋白H4乙酰化的鉴定，经酸性尿素-Triton凝胶电泳法分离得到三条蛋白质带，分别位于赖氨酸5、8、12和16位，分别为四乙酰化组蛋白H4、三乙酰化组蛋白H4和二乙酰化组蛋白H4。该方法可用于赖氨酸乙酰化的检测。
Posttranslational acetylation of proteins regulates many diverse functions; including DNA recognition, protein-protein interaction, and protein stability. The identification of enzymes that regulate protein acetylation has revealed broader use of this modification than was previously suspected. In this study, we describe a method for identifying protein acetylation at lysine residues by analysis of digested protein using HPLC/ESI-MS with a new modification-specific marker ion. Collision-induced dissociation with capillary or nano-LC/ESI-TOF-MS was used to obtain a fragment ion useful as a marker for acetylated lysine. Although the acetylated lysine immonium ion at m/z 143.1 has been used as a marker ion for detecting acetylated lysine, it can be confused with internal fragment ion in some peptides, producing false positive results. We have found a novel marker ion at m/z 126.1, which is a further fragment ion induced by the loss of NH3 from the acetylated lysine immonium ions at m/z 143.1: This novel. marker ion was found to be more specific and similar to9 times more sensitive than the immonium ion at m/z 143.1. In addition, no interfering ions for acetylated peptides were found in the extracted ion chromatogram at m/z 126.1. The utility of this method was demonstrated with acetylated cytochrome c as a model compound. After the modification was probed by the new marker ion, the acetylated lysine site was determined by the CID-MS spectrum. This method was applied to identify histone H4 acetylation in HeLa cells treated with trichostatin A. Three protein bands separated by acid-urea-Triton gel electrophoresis were confirmed as tetra, tri, and diacetylated histone H4 at lysines 5, 8, 12, and 16. This method may be useful for assaying for lysine acetylation, which is an important regulatory process for a range of biological functions.