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。次级项目的主要支助 而子项目的主要调查员可能是由其他来源提供的， 包括其它NIH来源。 为子项目列出的总成本可能 表示子项目使用的中心基础设施的估计数量， 而不是由NCRR赠款提供给子项目或子项目工作人员的直接资金。 棕榈酰化是蛋白质的脂质修饰，其中棕榈酰基通过硫酯键共价连接至半胱氨酸残基。棕榈酰化是可逆的和动态的。棕榈酰化和脱棕榈酰化之间的循环调节重要的细胞内事件。例如，膜相关的GT3-H-Ras通过调节棕榈酰化周转在不同的亚细胞区室之间穿梭，诱导其信号转导的变化。在这里，我们正在开发一种质谱法，直接检测棕榈酰化蛋白质/肽，并确定棕榈酰化位点。 这里选择的模型系统是合成的棕榈酰化肽GDIFNQVVPRC（palm）PR。肽的胰蛋白酶消化物的MALDI-TOF质谱显示棕榈酰基的部分损失，如CPR肽的存在所证明的。通过串联FTICR MS和串联TOF实验研究了棕榈酰化肽的裂解行为。选择三价带电荷的肽前体离子用于低能CID和ECD分析。低能量CID产生七个（潜在的十二个中的七个）残基间裂解。与以前的报告相反（J. Mass Spectrom. 2006年，41，229241），棕榈酰基保留在此处观察到的所有CID片段中。ECD产生一系列完整的残基间裂解，除了N-末端的脯氨酸残基。然而，一些ECD片段也以其脱棕榈酰化形式存在，尽管丰度低。还在MALDI-TOF/TOF仪器上对单电荷前体离子进行高能CID，其产生10个残基间裂解，并且没有棕榈酰基损失。 我们的研究结果表明，棕榈酰基团的损失可能发生在胰蛋白酶消化。因此，自上而下的方法可以优选用于蛋白质棕榈酰化的直接MS分析。棕榈酰化在CID和ECD下似乎都是稳定的，尽管ECD似乎是棕榈酰化位点鉴定的更好方法。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，使用自上而下和自下而上的方法，强调其在氧化应激下的凋亡途径中的作用。

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.