MASS SPECTROMETRY STUDY OF PROTEIN/PEPTIDE PALMITOYLATION

这个子项目是利用资源的许多研究子项目之一。 由NIH/NCRR资助的中心拨款提供。对子项目的主要支持 子项目的首席调查员可能是由其他来源提供的， 包括美国国立卫生研究院的其他来源。为子项目列出的总成本可能 表示该子项目使用的中心基础设施的估计数量， 不是由NCRR赠款提供给次级项目或次级项目工作人员的直接资金。 棕榈酰化是蛋白质的一种脂质修饰，通过硫代酯键将棕榈酰基共价连接到半胱氨酸残基上。棕榈酰化是可逆的和动态的。棕榈酸化和脱醛酸化之间的循环调节重要的细胞内事件。例如，膜相关的GTPase H-RAS通过调节棕榈酰化的周转，在不同的亚细胞间穿梭，引起其信号转导的变化。在这里，我们正在开发一种直接检测棕榈酰化蛋白质/肽并识别棕榈酰化位点的质谱学方法。 所选择的模型体系是人工合成的棕榈酰化肽GDIFNQVVPRC(Palm)PR。胰酶消化的MALDI-TOF质谱图显示，CPR多肽的存在使棕榈酰基部分丧失。用串联FTICR MS和串联TOF实验研究了棕榈酰化肽的裂解行为。选择三电荷肽前体离子进行低能CID和ECD分析。低能量的CID产生了7个(潜在的12个)残基间切割。与先前的报告相反(J.质谱学。2006年，41,229241)，在这里观察到的所有CID片段中都保留了棕榈酰基。ECD产生了一系列完整的残基间裂解，除了N-末端到脯氨酸残基。然而，一些ECD片段也以脱乙酰丝裂素的形式存在，尽管丰度很低。在MALDI-TOF/TOF仪器上对单电荷的前体离子进行高能CID，产生了10个残基间的裂解，没有棕榈酰基的损失。 我们的结果表明，在胰酶消化过程中可能会发生棕榈酰基的损失。因此，自上而下的方法可能是蛋白质棕榈酰化的直接MS分析的首选方法。棕榈酰化在CID和ECD下似乎都是稳定的，尽管ECD似乎是鉴定棕榈酰化位点的更好的方法。我们目前正在应用这种方法来跟踪人主动脉内皮细胞产生的H-RAS的棕榈酰化反应，包括自上而下和自下而上的方法，重点关注它在氧化应激下的细胞凋亡途径中的作用。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Palmitoylation is a lipid modification of proteins with the covalent attachment of a palmitoyl group to a cysteine residue through a thioester linkage. Palmitoylation is reversible and dynamic. Cycling between palmitoylation and depalmitoylation regulates important intracellular events. For example, membrane associated GTPase H-Ras shuttles among different subcellular compartments through regulation of palmitoylation turnover, inducing changes in its signaling transduction. Here we are developing a mass spectrometry method that directly detects palmitoylated proteins/peptides and identifies the palmitoylation sites. The model system chosen here is a synthetic palmitoylated peptide GDIFNQVVPRC(palm)PR. MALDI-TOF mass spectrum of the trypsin digest of the peptide showed partial loss of the palmitoyl group as evident by the presence of the CPR peptide. The fragmentation behavior of the palmitoylated peptide was investigated by tandem FTICR MS and tandem TOF experiments. Triply-charged peptide precursor ions were selected for low-energy CID and ECD analyses. Low-energy CID produced seven (out of the potential twelve) inter-residue cleavages. Contrary to a previous report (J. Mass Spectrom. 2006, 41, 229241), the palmitoyl group was retained in all CID fragments observed here. ECD produced a complete series of inter-residue cleavages except for that N-terminal to the proline residue. However, some ECD fragments were also present in their depalmitoylated form, albeit in low abundance. High-energy CID was also performed on the singly charged precursor ion on a MALDI-TOF/TOF instrument, which produced 10 inter-residue cleavages, and no palmitoyl group loss. Our results indicate that palmitoyl group losses could occur during trypsin digestion. Therefore, a top-down approach may be preferred for direct MS analysis of protein palmitoylation. Palmitoylation appeared to be stable under both CID and ECD, although ECD seemed to be the better method for palmitoylated site identification. We are currently applying this method to follow the palmitoylation in H-Ras produced by human aortic endothelial cells, using both top-down and bottom-up approaches, with emphasis on its role in apoptosis pathway under oxidative stress.