MASS SPECTROMETRY STUDY OF PROTEIN/PEPTIDE PALMITOYLATION

该子项目是利用资源的众多研究子项目之一 由 NIH/NCRR 资助的中心拨款提供。子项目的主要支持 并且子项目的主要研究者可能是由其他来源提供的， 包括其他 NIH 来源。 子项目可能列出的总成本 代表子项目使用的中心基础设施的估计数量， NCRR 赠款不直接向子项目或子项目工作人员提供资金。 棕榈酰化是蛋白质的脂质修饰，棕榈酰基通过硫酯键与半胱氨酸残基共价连接。棕榈酰化是可逆的、动态的。棕榈酰化和去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 GTPase 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方法，可以直接检测棕榈酰化蛋白质/肽并识别棕榈酰化位点。 这里选择的模型系统是合成的棕榈酰化肽 GDIFNQVVPRC(palm)PR。肽的胰蛋白酶消化的 MALDI-TOF 质谱显示棕榈酰基团部分丢失，CPR 肽的存在证明了这一点。通过串联 FTICR MS 和串联 TOF 实验研究了棕榈酰化肽的断裂行为。选择三电荷肽前体离子进行低能 CID 和 ECD 分析。低能 CID 产生了 7 个（可能有 12 个）残基间裂解。与之前的报告相反 (J. Mass Spectrom. 2006, 41, 229 241)，棕榈酰基保留在此处观察到的所有 CID 片段中。 ECD 产生了完整系列的残基间切割，除了脯氨酸残基的 N 端外。然而，一些 ECD 片段也以脱棕榈酰化形式存在，尽管丰度较低。还在 MALDI-TOF/TOF 仪器上对单电荷前体离子进行了高能 CID，产生 10 个残基间裂解，并且没有棕榈酰基损失。 我们的结果表明，胰蛋白酶消化过程中可能会发生棕榈酰基团损失。因此，对于蛋白质棕榈酰化的直接 MS 分析，自上而下的方法可能是首选。棕榈酰化在 CID 和 ECD 下似乎都很稳定，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中的棕榈酰化，使用自上而下和自下而上的方法，重点关注其在氧化应激下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.